Alpha-2 adrenergic receptor polymorphisms

ABSTRACT

The present invention includes polymorphisms in nucleic acids encoding the alpha-2B, alpha-2A, and alpha-2C adrenergic receptor and expressed alpha-2B, alpha-2A and alpha-2C adrenergic receptor molecule. The invention also pertains to methods and molecules for detecting such polymorphisms. The invention further pertains to the use of such molecules and methods in the diagnosis, prognosis, and treatment of diseases such as cardiovascular and central nervous system disease.

[0001] This application is a continuation-in-part and claims the benefitof the filing date of: U.S. application Ser. No. 09/551,744, filed Apr.17, 2000, entitled “Alpha-2C-Adrenergic Receptor Polymorphisms”, U.S.application Ser. No. 09/636,259, filed Aug. 10, 2000, entitled“Alpha-2A-Adrenergic Receptor Polymorphisms”, and U.S. application Ser.No. 09/692,077, filed Oct. 19, 2000, entitled “Alpha-2B-AdrenergicReceptor Polymorphisms” and International Application No:PCT/US01/12575, filed Apr. 17, 2001, entitled “Alpha-2 AdrenergicReceptor Polymorphisms”, these entire disclosures are herebyincorporated by reference into the present disclosure.

[0002] This invention was made, in part, with government support byNational Institutes of Health grants ES06096, and HL53436. The U.S.Government may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The invention relates to polymorphisms in the gene encoding analpha adrenergic receptor subtype. These polymorphisms result in alteredalpha-adrenergic receptor function and can cause or modify a diseaseand/or alter the response to pharmacologic treatment. More specifically,the present invention relates to polymorphisms in the alpha-2B, alpha-2Aand alpha-2C adrenergic receptor gene and the expressed alpha-2B,alpha-2A and the alpha-2C adrenergic receptor. The invention furtherrelates to methods and molecules for identifying one or morepolymorphisms in the alpha-2B, alpha-2A and alpha-2C adrenergic receptorgene and gene product. The present invention also provides methods ofdiagnosing, prognosing and treating individuals with diseases associatedwith one or more polymorphisms in the alpha-2B, alpha-2A and alpha-2Cadrenergic receptor.

BACKGROUND OF THE INVENTION

[0004] Alpha adrenergic receptors are plasma membrane receptors whichare located in the peripheral and central nervous systems throughout thebody. They are members of a diverse family of structurally relatedreceptors which contain seven putative helical domains and transducesignals by coupling to guanine nucleotide binding proteins (G-proteins).

[0005] The alpha adrenergic receptor family of adrenergic receptors (AR)consists of two groups: alpha-1 and alpha-2. Of the alpha-2 group, thereare three distinct subtypes denoted alpha-2A, alpha-2B and alpha-2C. Thesubtypes are derived from different genes, have different structures,unique distributions in the body, and specific pharmacologic properties.(Due to localization of the genes to human chromosomes 10, 2 and 4, thealpha-2A, alpha-2B, and alpha-2C receptors have sometimes been referredto as alpha-2C10, alpha-2C2 and alpha-2C4 receptors, respectively). Likeother adrenergic receptors, the alpha-2 receptors are activated byendogenous agonists such as epinephrine (adrenaline) and norepinephrine(noradrenaline), and synthetic agonists, which promote coupling toG-proteins that in turn alter effectors such as enzymes or channels.

[0006] The alpha-2 receptors couple to the G_(i) and G_(o) family ofG-proteins. Alpha-2 receptors modulate a number of effector pathways inthe cell: inhibition of adenylyl cyclase (decreases cAMP), stimulationof mitogen activated protein (MAP) kinase, stimulation of inositolphosphate accumulation, inhibition of voltage gated calcium channels andopening of potassium channels. (1,2). The alpha-2 receptors areexpressed on many cell-types in multiple organs in the body includingthose of the central and peripheral nervous systems.

[0007] There has been a considerable research effort to clone andsequence the alpha-2AR. For example, the gene encoding the alpha-2A,alpha-2B, alpha-2C subtypes has been cloned and sequenced. (Kobilka etal. Science 238, 650-656 (1987); Regan et al., Lomasney et al.Proc.Nat.Acad.Sci. 87, 5094-5098 (1994)).

[0008] Alpha-2BAR

[0009] Alpha-2BAR have a distinct pattern of expression within thebrain, liver, lung, and kidney, and recent studies using gene knockoutsin mice have shown that disruption of this receptor effects mouseviability, blood pressure responses to alpha-2-AR agonists, and thehypertensive response to salt loading. See (13);(14).

[0010] It is known that the alpha-2BAR undergoes short-term agonistpromoted desensitization(17). This desensitization is due tophosphorylation of the receptor, which evokes a partial uncoupling ofthe receptor from functional interaction with G_(i)/G_(o) (18, 19)).Such phosphorylation appears to be due to G protein coupled receptorkinases (GRKs), a family of serine/threonine kinases which phosphorylatethe agonist-occupied conformations of many G-protein coupled receptors(20). The phosphorylation process serves to finely regulate receptorfunction providing for rapid adaptation of the cell to its environment.Desensitization may also limit the therapeutic effectiveness ofadministered agonists. For the α_(2B)AR, phosphorylation ofserines/threonines in the third intracellular loop of the receptor isdependent on the presence of a stretch of acidic residues in the loopthat appears to establish the milieu for GRK function (18).

[0011] A polymorphism occurring in the gene encoding the alpha-2BAR hasbeen previously reported. This polymorphism has been described as adeletion of three glutamic acid residues in a highly acidic stretch ofamino acids in the third intracellular loop of the receptor. (21, 22).However, no pharmacologic studies have been carried out to determine ifthis polymorphism alters receptor function.

[0012] Given the importance of the alpha-2BAR in modulating a variety ofphysiological functions, there is a need in the art for improved methodsto identify polymorphisms and to correlate the identity of thesepolymorphisms with signaling functions of alpha-2BAR. The presentinvention addresses these needs and more by providing polynucleotide andamino acid polymorphisms, molecules, and methods for detecting,genotyping and haplotyping the polymorphisms in the alpha-2BAR. Thepresent invention is useful for determining an individual's risk fordeveloping a disease, assist the clinician in diagnosing and prognosingthe disease. The present invention also provides methods for selectingappropriate drug treatment based on the identity of such polymorphism.

[0013] Alpha-2AAR

[0014] Alpha-2AAR are the principal presynaptic inhibitory autoreceptorsof central and peripheral sympathetic nerves and inhibitneurotransmitter release in the brain and cardiac sympathetic nerves.(4). Such inhibition of neurotransmitter release in the brain is thebasis for the central hypotensive, sedative, anesthetic-sparing, andanalgesic responses of alpha-2AAR agonists (5,6). Indeed, alpha-2AARagonists such as clonidine and guanabenz are potent antihypertensiveagents which act via central presynaptic alpha-2AAR (7). The bloodpressure and other responses to alpha-2AAR agonists and antagonists,though, are subject to interindividual variation in the human population(7-9). Such variation, of course, can be due to genetic variation in thestructure of the receptor itself, its cognate G-proteins, the effectors,or downstream intracellular targets.

[0015] Of particular interest are physiologic and genetic studies whichsuggest that altered alpha-2AAR function can predispose individuals toessential hypertension ((7-9). Other physiologic functions of thealpha-2AAR are known. For example, the alpha-2AAR act to inhibit insulinsecretion by pancreatic beta-cells, contract vascular smooth muscle,inhibit lipolysis in adipocytes, modulate water and electrolyte flux inrenal cells, and aggregate platelets (3). Thus, like what has been shownwith beta-AR polymorphisms (12), potential polymorphisms of thealpha-2AAR may act as risk factors for disease, act to modify a givendisease, or alter the therapeutic response to agonists or antagonists.

[0016] Polymorphisms near the coding regions in the alpha-2A, alpha-2Cand dopamine β-hydroxylase (DBH) genes have been reported causingincreased levels of norepinephrine in children with attention-deficithyperactivity disorder (Comings et al. Clin Genet 55, 160-172 (1999)).Indeed, there have been several reports of non-coding regionpolymorphisms (i.e., in the 5′ and 3′ untranslated region) of the humanalpha-2A AR. One report has identified three SNPs in the coding region(Feng et al. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81, 405-410(1998). In this work, though, no pharmacologic studies were carried outto determine if these polymorphisms alter receptor function.

[0017] Given the importance of the alpha-2AAR in modulating a variety ofphysiological functions, there is a need in the art for improved methodsto identify polymorphisms and to correlate the identity of thesepolymorphisms with signaling functions of alpha-2AAR. The presentinvention addresses these needs and more by providing nucleic acid andamino acid polymorphisms, molecules, and methods for identifying thepolymorphisms in the alpha-2AAR. The present invention is useful fordetermining an individual's risk for developing a disease, assist theclinician in diagnosing and prognosing the disease. The presentinvention also provides methods for selecting appropriate drug treatmentbased on the identity of such polymorphism.

[0018] Alpha-2CAR

[0019] Alpha-2CAR plays specific roles in certain central nervous systemfunctions, such as for example, modulation of the acoustic startlereflex, prepulse inhibition, isolation induced aggregation, spatialworking memory, development of behavioral despair, body temperatureregulation, dopamine and serotonin metabolism, presynaptic control ofneurotransmitter release from cardiac sympathetic nerves, centralneurons, and postjunctional regulation of vascular tone Sallinen et al.Mol.Pharmacol. 51, 36-46 (1997); Sallinen et al. The Journal ofNeuroscience 18, 3035-3042 (1998); Tanila et al. European Journal ofNeuroscience 11, 599-603 (1999); Bjorklund et al. Mol Pharmacol 54,569-576 (1998); Hein et al. Nature 402, 181-184 (1999); Gavin et al.Naunyn Schmiedebergs Arch Pharmacol 355, 406-411 (1997); Sallinen et al.Mol Psychiatry 4, 443-452 (1999).

[0020] Polymorphisms near the coding regions in the alpha-2A, alpha-2Cand dopamine β-hydroxylase (DBH) genes have been reported causingincreased levels of norepinephrine in children with attention-deficithyperactivity disorder (Comings et al. Clin Genet 55, 160-172 (1999)).

[0021] To date polymorphisms occurring in nucleic acids encoding thealpha-2CAR receptor molecule and in the alpha-2C receptor have not beenreported.

[0022] Given the importance of the alpha-2CAR in modulating a variety ofphysiological functions, there is a need in the art for improved methodsto identify these polymorphisms and to correlate the identity of thesepolymorphisms with the physiological functions of alpha-2CAR. Thepresent invention addresses these needs and more by providing nucleicacid and amino acid polymorphisms, molecules, and methods foridentifying the polymorphisms in the alpha-2CAR. The present inventionis useful for determining an individual's risk for developing a diseaseand to diagnosis and prognosis the disease. The present invention alsoprovides methods for selecting appropriate drug treatment based on theidentity of such polymorphisms.

SUMMARY OF THE INVENTION

[0023] The present invention provides methods, molecules, kits, andprimers useful for detecting one or more polymorphic sites inpolynucleotides encoding the alpha-2B, alpha-2A or alpha-2C adrenergicreceptor gene and gene products.

[0024] In some embodiments, the present invention provides polymorphismsin nucleic acids encoding the alpha-2B, alpha-2A, and alpha-2Cadrenergic receptor and expressed alpha-2B, alpha-2A and alpha-2Cadrenergic receptor molecule.

[0025] In other embodiments, the present invention provides moleculesand methods to diagnosis, prognosis, and treat diseases such ascardiovascular and central nervous system disease that are associatedwith the alpha-2B, alpha-2A or alpha-2C adrenergic receptor gene and/orgene products.

[0026] For a better understanding of the present invention together withother and further advantages and embodiments, reference is made to thefollowing description taken in conjunction with the examples, the scopeof which is set forth in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Preferred embodiments of the invention have been chosen forpurposes of illustration and description, but are not intended in anyway to restrict the scope of the invention. The preferred embodiments ofcertain aspects of the invention are shown in the accompanying figures,wherein:

[0028]FIG. 1 illustrates identification of the human alpha-2BAR variant.Shown in Panels A and B are representative automated sequencechromatograms identifying a deletion of the nucleotides GAAGAGGAG (SEQID NO: 3). Panel C illustrates a rapid screening technique thatidentifies homozygous and heterozygous PCR products by size.

[0029]FIG. 2 illustrates localization of the expressed alpha-2BARpolymorphism. Shown is the fifth and sixth transmembrane spanning domain(TMD) and the third intracellular loop of the receptor.

[0030]FIG. 3 graphically illustrates that the alpha-2BAR with deletionin amino acids 301 to 303 (Del301-303) of SEQ ID NO: 8, fails to undergoshort-term agonist-promoted desensitization. Cells in culture expressingthe two receptors were exposed to vehicle or 10 μM norepinephrine for 30minutes at 37° C., washed extensively, membranes prepared and adenylylcyclase activities determined as described in the examples. Panels A andB show results of full dose-response studies, which reveal that whilethe wild-type receptor (SEQ ID NO: 7) undergoes desensitizationmanifested as a rightward shift in the curve, the Del301-303 mutant doesnot. Panel C shows the percent inhibition of adenylyl cyclase at asubmaximal concentration of norepinephrine in the assay (the EC₅₀ of thecontrol membranes) for both wild-type and mutant conditions, indicatingan ˜54% desensitization of wild-type alpha-2BAR. The Del301-303 failedto display such desensitization. Results are from four independentexperiments. See also Table 9. *=p<0.05 compared to control.

[0031]FIG. 4 illustrates that alpha-2BAR with deletion in amino acids301 to 303 (Del301-303) has impaired agonist-promoted phosphorylation.Cells co-expressing each receptor and GRK2 were incubated with³²P-orthophosphate, exposed to 10 μM norepinephrine for 15 minutes, andreceptor purified by immunoprecipitation as described in the examples.Shown is an autoradiogram from a single experiment representative offour performed.

[0032]FIG. 5 illustrates sequence variation of the human alpha-2AAR atnucleotide position 753. Shown are sequence electropherograms (sensestrand) of PCR products amplified from individuals homozygous for thewild-type alpha-2AAR and Lys251 receptor (Panels A and B), and aheterozygous individual (Panel C) as described below. The cytosine inthe indicated position of codon 251 results in an Asn, whereas a guanineencodes for Lys. Panel D shows agarose gel of PCR products fromhomozygous wild-type (Asn), heterozygous (Asn/Lys), and homozygouspolymorphic (Lys) individuals digested with Sty I. The C to Gtransversion at nucleotide 753 creates a unique Sty I site that resultsin partial and complete digestion of a 556 bp fragment amplified fromLys251 heterozygous and homozygous individuals, respectively.

[0033]FIG. 6 illustrates the location of the Lys251 alpha-2AARpolymorphism and alignment of flanking amino acid residues of the thirdintracellular loop from various species. The locations of the Lys251amino acid polymorphism in the third intracellular loop as well as twosynonymous SNPs (single nucleotide polymorphisms) are indicated.Alignment of alpha-2AAR amino acid sequence from various species showsthat this region is highly conserved and that Asn at position 251 isinvariant in all mammalian species reported to date except for humanswhere we have noted the Lys polymorphism. Amino acids in the mid-portionof the third intracellular loop are represented as solid dots forconvenience.

[0034]FIG. 7 is a graphic illustration of the coupling of the wild-typeAsn251 and polymorphic Lys251 alpha-2AARs to the inhibition of adenylylcyclase. Membranes from CHO cells were prepared and adenylyl cyclaseactivities determined as described below in the presence of 5.0 μMforskolin and the indicated concentrations of the full agonistepinephrine (Graph A) and the partial agonist oxymetazoline (Graph B).Results as shown are the percent inhibition of forskolin stimulatedactivities from clones at matched levels of expression (˜2500 fmol/mg)from 5 individual experiments each (*indicates p<0.05 for the maximalinhibition compared to wild-type for both agonists).

[0035]FIG. 8 is a bar graph illustration of wild-type Asn251 andpolymorphic Lys251 alpha-2AAR promoted [³⁵S]GTPγS binding in response tofull and partial agonists. Binding of [³⁵S]GTPγS was measured inmembranes from COS-7 cells transiently coexpressing the wild-type andLys251 alpha-2AAR and G_(iα2) as described below. Assays were carriedout using 10 μM of the agonists UK 14304, epinephrine, norepinephrine,BHT-933, clonidine, guanabenz, and oxymetazoline. Results are shown as %increase over basal levels from three or more experiments.

[0036]FIG. 9 illustrates stimulation of MAP kinase by wild-type andLys251 alpha-2AARs. Phosphorylation of MAP kinase was determined in CHOcells by quantitative immunoblotting with enhanced chemifluorescenceusing antibodies specific for phosphorylated Erk 1/2. The same blotswere stripped and reprobed for total MAP kinase expression, which wasnot significantly different between the two cell lines (Panel A). Cellswere studied after incubation with carrier (basal), 10 μM epinephrine,or 1 unit/ml thrombin. Results are shown as the fold-stimulation overbasal levels (Panel B). The * indicates p<0.05 compared to the wild-typeresponse (n=3 experiments).

[0037]FIG. 10 is a bar graph illustration of stimulation of inositolphosphate accumulation by wild-type Asn251 and polymorphic Lys251alpha-2AARs. Total inositol phosphate production in intact CHO cells wasmeasured as described below in response to a 5 minute exposure to 10 μMepinephrine. Results are from five experiments.

[0038]FIG. 11 illustrates sequence variation of the human alpha-2CAR atnucleotides 964-975. Shown are automated sequencing chromatograms (sensestrand) from individuals homozygous for the wild-type alpha-2CAR (PanelA) and Del322-325 polymorphism (Panel B). The underlined bases in Arepresent the nucleotides that were found to be deleted in thepolymorphic sequence (arrow in B). Panel C shows agarose gel of PCRproducts from wild-type (WT) homozygous (384 bp), Del322-325 homozygous(372 bp), and heterozygous individuals digested with Nci 1. Wild-typereceptor provides for the bands at the indicated molecular sizes (twoproducts of 6 and 1 bp are not shown). The loss of one of the six Nci 1sites due to the polymorphism results in a unique product of 111 bp andloss of the 82 and 41 bp products. Heterozygotes have all six fragments.

[0039]FIG. 12 illustrates the localization of the alpha-2CARpolymorphism. Shown is the amino acid sequence and the proposed membranetopology of the fifth and sixth transmembrane spanning domains and thethird intracellular loop. The polymorphism results in the loss ofGly-Ala-Gly-Pro at the indicated position. The third intracellular loopis shown in a compact form for illustrative purposes and is not intendedto represent known secondary structure.

[0040]FIG. 13 is a graphic illustration of the coupling of wild-type andmutant with deletions in amino acids 322-325 (Del322-325) alpha-2CARs tothe inhibition of adenylyl cyclase. Membranes from CHO cells wereprepared and adenylyl cyclase activities determined in the presence of5.0 μM forskolin and the indicated concentrations of epinephrine.Results are shown as the percent inhibition of forskolin stimulatedactivities. (For all cell lines the fold stimulation by forskolin was˜10 fold over basal levels). Panel A shows results from two cell linesexpressing the wild-type and Del322-325 receptors at ˜1380±140 and˜1080±160 fmol/mg. Panel B shows results from lower levels of expressionin two other cell lines with densities of 565±69 and 520±51 fmol/mg,respectively. Results are from 5 experiments. *, p<0.001 for the maximalinhibition compared to wild-type.

[0041]FIG. 14 is a bar graph illustration of stimulation of inositolphosphate accumulation by wild-type and Del322-325 alpha-2CARs. Totalinositol phosphate production in intact CHO cells was measured inresponse to 5 minute exposure to 10 μM epinephrine. Receptor expressionwas 806±140 and 733±113 fmol/mg, respectively, for these experiments. *,p<0.005 compared to wild-type response (n=4 experiments).

[0042]FIG. 15 illustrates stimulation of MAP kinase by wild-type andDel322-325 alpha-2CARs. Phosphorylation of MAP kinase was determined inCHO cells by quantitative immunoblotting with enhanced chemifluorescenceusing antibodies specific for phosphorylated Erk1/2. The same blots werestripped and reprobed for total MAP kinase expression, which was notsignificantly different between the two cell lines (Panel A). Cells werestudied after incubation with carrier (basal), 10 μM epinephrine or 1unit/ml thrombin. Results are depicted as the fold-stimulation overbasal normalized to the wild-type response (Panel B) and the percent ofthe thrombin response (Panel C). *, p<0.005 compared to wild-typeresponse (n=5 experiments).

DETAILED DESCRIPTION OF THE INVENTION

[0043] Alpha-2BAR, Alpha-2AAR and Alpha-2CAR Functions

[0044] The alpha-2 adrenergic receptors are localized at the cellmembrane and serves as receptors for endogenous catecholamine agonistsi.e., epinephrine and norepinephrine, and synthetic agonists andantagonists. Upon binding of the agonist, the receptors stabilize in aconformation that favors contact with all activation of certainheterotrimeric G proteins. These include G_(i1), G_(i2), G_(i3) andG_(o). The G_(i) G protein alpha subunits serve to decrease the activityof the enzyme adenylyl cyclase, which lowers the intracellular levels ofcAMP (a classic second messenger). The alpha subunits, and/or thebeta-gamma subunits of these G proteins also act to activate MAP kinase,open potassium channels, inhibit voltage gated calcium channels, andstimulate inositol phosphate accumulation. The physiologic consequencesof the initiation of these events include inhibition of neurotransmitterrelease from central and peripheral noradrenergic neurons.

[0045] Alpha-2BARs are expressed in the brain, liver, lung, and kidney.Studies using gene knockouts in mice have shown that disruption of thisreceptor effects mouse viability, blood pressure responses to alpha-2-ARagonists, and the hypertensive response to salt loading. Alpha-2BAR whenstimulated cause vasoconstriction.

[0046] Alpha-2BARs undergoes short-term agonist promoteddesensitization. This desensitization is due to phosphorylation of thereceptor, which evokes a partial uncoupling of the receptor fromfunctional interaction with G_(i)/G_(o). Such phosphorylation appears tobe due to GRKs, a family of serine/threonine kinases which phosphorylatethe agonist-occupied conformations of many G-protein coupled receptors.Desensitization may also limit the therapeutic effectiveness ofadministered agonists. For the alpha-2BAR, phosphorylation ofserines/threonines in the third intracellular loop of the receptor isdependent on the presence of a stretch of acidic residues in the loopthat appears to establish the milieu for GRK function (18).

[0047] The alpha-2A has been localized in brain, blood vessels, heart,lung, skeletal muscle, pancreas, kidney, prostate, ileum, jejunum,spleen, adrenal gland and spinal cord (Eason et al. MolecularPharmacology 44, 70-75 (1993); Zeng et al. Mol Brain Res 10, 219-225(1991).

[0048] Alpha-2AARs are widely expressed and participate in a broadspectrum of physiologic functions including metabolic, cardiac,vascular, and central and peripheral nervous systems, via pre-synapticand post-synaptic mechanisms. At peripheral sites, alpha-2AARs act toinhibit insulin secretion by pancreatic beta-cells, contract vascularsmooth muscle, inhibit lipolysis in adipocytes, modulate water andelectrolyte flux in renal cells, and aggregate platelets (3). Asdiscussed above, the alpha-2AAR is the principal presynaptic inhibitoryautoreceptor of central and peripheral sympathetic nerves (4). Suchinhibition of neurotransmitter release in the brain is the basis for thecentral hypotensive, sedative, anesthetic-sparing, and analgesicresponses of alpha-2AAR agonists (5,6).

[0049] Alpha-2AAR agonists such as clonidine and guanabenz are potentantihypertensive agents which act via central presynaptic alpha-2AARs(7). The blood pressure and other responses to alpha-2AAR agonists andantagonists, though, are subject to interindividual variation in thehuman population (7-9). Such variation can be due to genetic variationin the structure of the receptor itself, its cognate G-proteins, theeffectors, or downstream intracellular targets. Of particular interestare physiologic and genetic studies which suggest that alteredalpha-2AAR function can predispose individuals to essentialhypertension. See for example,(7-9) (10,11). Thus, like what has beenshown with beta-AR polymorphisms (12), potential polymorphisms of thealpha-2AAR can act as risk factors for disease, act to modify a givendisease, or alter the therapeutic response to agonists or antagonists.

[0050] The alpha-2C has been localized in brain, blood vessels, heart,lung, skeletal muscle, pancreas, kidney, prostate, ileum, jejunum,spleen, adrenal gland and spinal cord (Eason et al. MolecularPharmacology 44, 70-75 (1993); Zeng et al. Mol Brain Res 10, 219-225(1991).Alpha-2CAR plays specific roles in certain central nervous systemfunctions, such as for example, modulation of the acoustic startlereflex, prepulse inhibition, isolation induced aggression, spatialworking memory, development of behavioral despair, body temperatureregulation, dopamine and serotonin metabolism, presynaptic control ofneurotransmitter release from cardiac sympathetic nerves and centralneurons, and postjunctional regulation of vascular tone Sallinen et al.Mol.Pharmacol. 51, 36-46 (1997); Sallinen et al. The Journal ofNeuroscience 18, 3035-3042 (1998); Tanila et al. European Journal ofNeuroscience 11, 599-603 (1999); Bjorklund et al. Mol Pharmacol 54,569-576 (1998); Hein et al. Nature 402, 181-184 (1999); Gavin et al.Naunyn Schmiedebergs Arch Pharmacol 355, 406-411 (1997); Sallinen et al.Mol Psychiatry 4, 443-452 (1999).

[0051] Since most organs have innervation by these neurons, theactivities of the alpha-2A, the alpha-2B and the alpha-2C receptors canalter processes in many organ systems. Of particular therapeuticinterest has been the development of highly subtype-specific alpha-2agonists and antagonists. Such compounds, then, can selectively block oractivate one subtype, such as the alpha-2B, without affecting theothers. This would provide for highly specific responses withoutside-effects from activating the other subtypes.

[0052] Alpha-2B, alpha-2A and alpha-2C adrenergic receptor moleculefunction or activity can be measured by methods known in the art. Someexamples of such measurement include radio-ligand binding to thealpha-2B, alpha-2A or alpha-2C adrenergic receptor molecule by anagonist or antagonist, receptor-G protein binding, stimulation orinhibition of adenyly cyclase, MAP kinase, phosphorylation or inositolphosphate (IP3). However, the polymorphisms of the present invention(discussed below) alter their respective alpha-2 adrenergic receptormolecule function or activity.

[0053] In one embodiment of the present invention, the DEL301-303polymorphism showed depressed phosphorylation resulting in loss ofshort-term agonist-promoted receptor desensitization in the alpha-2BARmolecule. The DEL301-303 polymorphism also showed altered or decreasedreceptor coupling.

[0054] Alpha-2 Adrenergic Receptor Diseases

[0055] Alpha-adrenergic receptors play an important role in regulating avariety of physiological functions because of their distribution in manyorgans of the body and the brain. Thus, dysfunctional alpha-2B, alpha-2Aor alpha-2C receptors can predispose to, or modify, a number of diseasesor alter response to therapy. The present invention stems in part fromthe recognition that certain polymorphisms in the alpha-2BAR, alpha-2AARor alpha-2CAR result in receptor molecules with altered functions. Thesealtered functions put an individual at risk for developing diseasesassociated with the alpha-2BAR, alpha-2AAR or the alpha-2ACR.

[0056] As used herein, “disease” includes but is not limited to anycondition manifested as one or more physical and/or psychologicalsymptoms for which treatment is desirable, and includes previously andnewly identified diseases and other disorders. Such diseases includecardiovascular diseases such as hypertension, hypotension, congestiveheart failure, arrhythmias, stroke, myocardial infarction, neurogenicand obstructive peripheral vascular disease, ischemia-reperfusion damageand intermittent claudication, migraine, metabolic rate and combinationsthereof. Central nervous systems (CNS) diseases are also contemplated bythe present invention. Some examples of CNS diseases includeParkinsonism, Alzheimers, attention deficit disorder, hyperreactivity,anxiety, manic-depression and combinations thereof. Since the alpha-2B,alpha-2A and alpha-2C control certain central nervous system andperipheral functions as discussed above, dysfunctional polymorphisms arelikely to be important in as of yet unclassified disorders of memory andbehavior.

[0057] In one embodiment, the present invention includes methods ofdetermining the risk an individual has for developing a disease.Alternatively, the present invention can be used to diagnose or prognosean individual with a disease. For example, a polymorphic site in thepolynucleotide encoding the mutant alpha-2BAR identified as SEQ ID NO:2, such as for example, nucleotide positions 901 to 909 can be detected.This polymorphic site corresponds to GAGGAGGAG (SEQ ID NO: 4). Thus, themutant receptor has a deletion of nine nucleotides (DEL901-909)GAAGAGGAG (SEQ ID NO: 3) when compared to the polynucleotide encodingthe wild-type alpha-2BAR (IN901-909). This exemplified polymorphismresults in amino acid deletions at positions 301 to 303 of the mutantalpha-2B adrenergic receptor molecule resulting in the mutant receptoridentified as SEQ ID NO: 8. More particularly, the preferredpolymorphism results in a deletion of 3 glutamic acids at amino acidpositions 301 to 303 (DEL 301-303) of the alpha-2B adrenergic receptormolecule resulting in a receptor with decreased alpha-agonist function.Such polymorphism can be correlated to increasing an individual's riskfor developing a disease, or can be used to determine a diagnosis orprognosis for the disease.

[0058] In another embodiment of the present invention, a polymorphicsite in SEQ ID NO: 1, such as for example, nucleotide position 901 to909 can be detected. This polymorphic site corresponds to (IN901-909)GAAGAGGAG (SEQ ID NO: 3) that is an insertion of these nine nucleotidescompared to the polynucleotide encoding the mutant alpha-2BAR. Thisexemplified polymorphism results in amino acid insertion at positions301 to 303 (IN301-303) of the alpha-2B adrenergic receptor moleculeresulting in the wild-type receptor identified as SEQ ID NO: 7. Moreparticularly, the preferred polymorphism results in an insertion of 3glutamic acids at amino acid positions 301 to 303 of the alpha-2Badrenergic receptor molecule resulting in a receptor with increasedalpha-agonist function and increased agonist-promoted desensitization.Such polymorphism can be correlated to decreasing an individual's riskfor developing a disease, or can be used to determine a diagnosis orprognosis for the disease.

[0059] For the alpha-2AAR, a polymorphic site in SEQ ID NO: 24, such asfor example, nucleotide position 753 can be detected. This polymorphicsite corresponds to a cytosine in nucleic acids encoding the alpha-2AAR.This exemplified polymorphism results in asparagine at amino acidposition 251 of SEQ ID NO: 26 of the alpha-2A adrenergic receptormolecule resulting in a receptor with decreased alpha-agonist function.Such polymorphism can be correlated to increasing an individual's riskfor developing a disease, or can be used to determine a diagnosis orprognosis for the disease.

[0060] In another embodiment of the present invention, a polymorphicsite in SEQ ID NO: 25, such as for example, nucleotide position 753 canbe detected. This polymorphic site corresponds to a guanine in nucleicacids encoding the alpha-2AAR. This exemplified polymorphism results inlysine at amino acid position 251 of SEQ ID NO: 27 of the alpha-2Aadrenergic receptor molecule resulting in a receptor with increasedalpha-agonist function. Such polymorphism can be correlated toincreasing an individual's risk for developing a disease, or can be usedto determine a diagnosis or prognosis for the disease.

[0061] For the alpha-2CAR, a polymorphic site in SEQ ID NO: 42, such asfor example, SEQ ID NO: 43 which corresponds to a twelve nucleotidedeletion in nucleic acids is detected. This exemplified polymorphismresults in the deletion of amino acids 322-325 of alpha-2C adrenergicreceptor molecule resulting in a defective receptor.

[0062] As used herein, “diagnosis” includes determining the nature andcause of the disease, based on signs and symptoms of the disease andlaboratory finding. One such laboratory finding is the identification ofat least one polymorphism in nucleic acids encoding the alpha-2BAR, thealpha-2AAR or the alpha-2CAR. Prognosis of a disease includesdetermining the probable clinical course and outcome of the disease.Increased risk for the disease includes an individual's propensity orprobability for developing the disease.

[0063] The terms “correlate the polymorphic site with a disease”includes associating the polymorphism which occurs at a higher allelicfrequency or rate in individuals with the disease than individualswithout the disease. Correlation of the disease with the polymorphismcan be accomplished by bio-statistical methods known in the art, such asfor example, by Chi-squared tests or other methods described by L. D.Fisher and G. vanBelle, Biostatistics: A Methodology for the HealthSciences, Wiley-Interscience (New York) 1993.

[0064] Preferably, the identity of at least one polymorphic site in analpha-2B, alpha-2A or alpha-2C adrenergic receptor molecule isdetermined. Generally, in performing the methods of the presentinvention, the identity of more than one polymorphic site is determined.As used herein a polymorphic site includes one or more nucleotidedeletions (DEL), insertions (IN), or base changes at a particular sitein a nucleic acid sequence. In some preferred embodiments, the identityof between about two and about six polymorphic sites is determined,though the identification of other numbers of sites is also possible.Most preferably, the polymorphisms and molecules of the presentinvention are utilized in determining the identity of at least onepolymorphic site of the alpha-2BAR, alpha-2AAR or alpha-2CAR moleculeand using that identity as a predictor of increased risk for developinga disease. The type of polymorphism present can also dictate theappropriate drug selection. In other embodiments, the polymorphisms andmolecules of the present are used for diagnosing or prognosing anindividual with a disease associated with an alpha-2BAR, alpha-2AAR oralpha-2CAR molecule.

[0065] Alpha-2 Adrenergic Receptor Polymorphisms

[0066] The particular gene sequences of interest to the presentinvention comprise “mutations” or “polymorphisms” in the genes encodingthe alpha-2B, alpha-2A and alpha-2C adrenergic receptor. The genomes ofanimals and plants naturally undergo spontaneous mutation in the courseof their continuing evolution (J. F. Gusella (1986) Ann. Rev. Biochem.55:831-854). These mutations may be in the form of deletions (DEL),insertions (IN), or base changes at a particular site in a nucleic acidsequence. This altered sequence and the initial sequence may co-exist ina species' population. In some instances, these changes confer neitheran advantage or a disadvantage to the species and multiple alleles ofthe sequence may be in stable or quasi-stable equilibrium. In someinstances, however, these sequence changes will confer a survival orevolutionary advantage to the species, and accordingly, the alteredallele may eventually (i.e. over evolutionary time) be incorporated intothe genome of many or most members of that species. In other instances,the altered sequence confers a disadvantage to the species, as where themutation causes or predisposes an individual to a genetic disease. Asused herein, the terms “mutation” or “polymorphism” refer to thecondition in which there is a variation in the DNA sequence between somemembers of a species. Typically, the term “mutation” is used to denote avariation that is uncommon (less than 1%), a cause of a rare disease,and that results in a gene that encodes a non-functioning protein or aprotein with a substantially altered or reduced function. Such mutationsor polymorphisms include, but are not limited to, single nucleotidepolymorphisms (SNPs), one or more base deletions, and one or more baseinsertions.

[0067] Polymorphisms may be synonymous or nonsynonymous. Synonymouspolymorphisms when present in the coding region typically do not resultin an amino acid change. Nonsynonymous polymorphism when present in thecoding region alter one or more codons resulting in an amino acidreplacement in the amino acid chain. Such mutations and polymorphismsmay be either heterozygous or homozygous within an individual.Homozygous individuals have identical alleles at one or morecorresponding loci on homologous chromosomes. While heterozygousindividuals have two different alleles at one or more corresponding locion homologous chromosomes. A polymorphism is thus said to be “allelic,”in that, due to the existence of the polymorphism, some members of aspecies carry a gene with one sequence (e.g., the original or wild-type“allele”), whereas other members may have an altered sequence (e.g., thevariant or, mutant “allele”). In the simplest case, only one mutatedvariant of the sequence may exist, and the polymorphism is said to bediallelic. For example, if the two alleles at a locus areindistinguishable in their effects on the organism, then the individualis said to be homozygous at the locus under consideration. If the twoalleles at a locus are distinguishable because of their differingeffects on the organism, then the individual is said to be heterozygousat the locus. In the present application, typographically, alleles aredistinguished +and −. Using these symbols, homozygous individuals are+/+, or −/−. Heterozygous individuals are +/−. The occurrence ofalternative mutations can give rise to triallelic and tetra-allelicpolymorphisms, etc. An allele may be referred to by the nucleotide(s)that comprise the mutation.

[0068] Alpha 2-BAR Polymorphisms

[0069] The wild-type gene encoding the third intracellular loop of thehuman alpha-2B receptor molecule is disclosed in GenBank Accession No.#AF009500, the entire disclosure is herein incorporated by reference. Asused herein, the term “gene” includes a segment of DNA that contains allthe information for the regulated biosynthesis of an RNA product,including promoters, exons, introns, and other untranslated regions thatcontrol expression.

[0070] The terms “alpha-2B-adrenergic receptor polymorphism” or“alpha-2BAR polymorphism”, are terms of art and refer to at least onepolymorphic site in the polynucleotide or amino acid sequence of analpha-2B adrenergic receptor gene or gene product. For purposes of thepresent application, the wild-type polynucleotide encoding thealpha-2B-adrenergic receptor is designated SEQ ID NO: 1 and thewild-type gene product comprising the alpha-2B-adrenergic receptormolecule, is designated amino acid SEQ ID NO: 7.

[0071] Those in the art will readily recognize that nucleic acidmolecules may be double-stranded molecules and that reference to aparticular site on one strand refers, as well, to the corresponding siteon a complementary strand. Thus, in defining a polymorphic site,reference to an adenine, a thymine (uridine), a cytosine, or a guanineat a particular site on the plus (sense) strand of a nucleic acidmolecule is also intended to include the thymine (uridine), adenine,guanine, or cytosine (respectively) at the corresponding site on a minus(antisense) strand of a complementary strand of a nucleic acid molecule.Thus, reference may be made to either strand and still comprise the samepolymorphic site and an oligonucleotide may be designed to hybridize toeither strand. Throughout this specification, in identifying apolymorphic site, reference is made to the sense strand, only for thepurpose of convenience.

[0072] Preferred polymorphisms of the present invention occur in thegene encoding the alpha-2B adrenergic receptor molecule identified asSEQ ID NO: 1 or 2 or fragments thereof or complements thereof.

[0073] For the purposes of identifying the location of at least onepolymorphism or polymorphic site, the first nucleotide of the startcodon of the coding region (the adenine of the ATG in a DNA molecule andthe adenine of the AUG in an RNA molecule) of the alpha-2BAR gene isconsidered nucleotide “1.” This corresponds to nucleotide 1 of SEQ IDNO: 1 or 2. The end of the coding region corresponds to adenine atposition 1353 for SEQ ID NO: 1. The end of the coding region correspondsto adenine at position 1344 for SEQ ID NO: 2. According to the presentinvention, polymorphisms can occur any where in the coding regionidentified as SEQ ID NO: 1 or 2.

[0074] For example, the polymorphism occurring in the polynucleotideencoding the wild-type alpha-2BAR molecule (identified as SEQ ID NO: 1)is a nine nucleotide base insertion (IN901-909) at nucleotide positions901 to 909 of SEQ ID NO: 1. This nine nucleotide base insertion isidentified as GAAGAGGAG (SEQ ID NO: 3) and is a polymorphic site orfragment (FIG. 1A) of SEQ ID NO: 1. A complement to this polymorphicsite includes CTTCTCCTC (SEQ ID NO: 5).

[0075] In another embodiment of the present invention, at least onepolymorphic site has been identified in the polynucleotide encoding themutant alpha-2BAR identified as SEQ ID NO: 2. This polymorphic site is anine nucleotide deletion at nucleotide positions 901 to 909(DEL901-909). This polymorphic site shifts GAGGAGGAG (SEQ ID NO: 4) intonucleotide positions 901 to 909. Thus, the polynucleotide encoding themutant receptor has a deletion of nine nucleotides (FIG. 1B) GAAGAGGAG(SEQ ID NO: 3) when compared to the polynucleotide encoding thewild-type alpha-2BAR (identified as SEQ ID NO: 1). A compliment to theGAGGAGGAG (SEQ ID NO: 4) polymorphic site includes CTCCTCCTC (SEQ ID NO:6).

[0076] An insertion or deletion polymorphism can change the exactposition of at least one polymorphic site with respect to thepolynucleotide encoding the alpha2-BAR identified as SEQ ID NO: 1 or 2.The present invention includes polymorphic sites occurring downstream ofthe IN/DEL901-909 polymorphic site. For example, detecting one or moresingle nucleotide polymorphisms (SNP) such as G at nucleotide position915 and/or G at 951, will indicate the I901-909 polymorphism.Alternately, the end of the coding region of the polynucleotide can beprobed to determine the longer polynucleotide indicating the IN901-909polymorphism. For example, if a G SNP is detected at nucleotide position1345, this indicates, the IN901-909 polymorphism of SEQ ID NO: 1, sincethe mutant SEQ ID NO: 2 does not have this nucleotide position in thecoding region. Thus, the IN/DEL901-909 polymorphic site can indirectlybe detected by the nucleotide shift resulting from the insertion ordeletion.

[0077] As used herein, “fragments of the polynucleotide encoding thealpha2-BAR” include less than the entire nucleotide sequence of SEQ IDNO: 1 or 2. The fragments comprise the polymorphic site or areassociated with the polymorphic site. Preferred fragments comprise theIN901-909 or DEL901-909 polymorphic site. In order for a nucleic acidsequence to be a fragment, it must be readily identifiable by themolecular techniques as discussed such as with nucleic acid probes.

[0078] The polymorphisms of the present invention can occur in thetranslated alpha-2B adrenergic receptor molecule as well. For example,the first amino acid of the translated protein product or gene product(the methionine) is considered amino acid “1” in the wild-type alpha-2Badrenergic receptor molecule designated amino acid SEQ ID NO: 7. The endof the receptor corresponds to tryptophan at amino acid position 450 forSEQ ID NO: 7. Polymorphisms can occur anywhere in SEQ ID NO: 7. Thewild-type alpha-2B adrenergic receptor molecule (FIG. 2) comprises aninsertion of 3 glutamic acids at amino acid positions 301 to 303(IN301-303) of the alpha-2B adrenergic receptor molecule designated EEE(SEQ ID NO: 11). Thus, in the stretch of amino acids at positions294-309 identified as EDEAEEEEEEEEEEEE (SEQ ID NO: 9), there is aninsertion of three additional glutamic acids when compared to the mutantalpha-2B adrenergic receptor molecule. Accordingly, EDEAEEEEEEEEEEEE(SEQ ID NO: 9) and EEE (SEQ ID NO: 11) are examples of polymorphic sitesoccurring in SEQ ID NO: 7.

[0079] In another embodiment of the present invention, SEQ ID NO: 8comprises the entire mutant amino acid sequence of alpha-2B adrenergicreceptor molecule with deletion of EEE at amino acid positions 301-303(DEL301-303) (shown in FIG. 2). The first amino acid of the translatedprotein product or gene product (the methionine) is considered aminoacid “1” in the mutant alpha-2B adrenergic receptor molecule designatedamino acid SEQ ID NO: 8. The end of the receptor corresponds totryptophan at amino acid position 447 for SEQ ID NO: 8. Polymorphismscan occur anywhere in the amino acid sequence designated SEQ ID NO: 8.For example, the mutant alpha-2B adrenergic receptor molecule comprisesa deletion of EEE (SEQ ID NO: 11) or 3 glutamic acids in the mutantalpha-2B adrenergic receptor molecule. Thus, in the stretch of aminoacids at positions 294-306 identified as EDEAEEEEEEEEE (SEQ ID NO: 10).There is a deletion of three glutamic acids when compared to thewild-type alpha-2B adrenergic receptor molecule. Accordingly,EDEAEEEEEEEEEEEE (SEQ ID NO: 10) and EEE (SEQ ID NO: 11) are examples ofpolymorphic sites occurring in SEQ ID NO: 8.

[0080] Since the mutant alpha-2B adrenergic receptor molecule hasDEL301-303, the rest of the molecule shifts causing amino acids 307, 308and 309 to have CEP at these positions in the mutant receptor. Thus,another polymorphic site is CEP (SEQ ID NO: 12) at amino acid positions307-309 of SEQ ID NO: 8, individually and /or collectively, thesepositions represent polymorphic sites when compared to the wild-typereceptor. Alternatively, the mutant alpha-2B adrenergic receptormolecule lacks amino acids positions 448, 449 and 450. Thus, these arealso polymorphic sites.

[0081] For example, an insertion or deletion polymorphisms can changethe exact position of at least one polymorphic site with respect to theamino sequence of the alpha-2-BAR identified as SEQ ID NO: 7 or 8. Thepresent invention includes one or more polymorphic sites occurringdownstream of the IN/DEL301-303 polymorphic site. For example, detectingR at amino position 340 and/or R at 438, will indicate the IN301-303polymorphism. Alternately, the end of the coding region of thepolynucleotide can be probed to determine the longer chain of aminoacids indicating the IN301-303 polymorphism. For example, if a T isdetected at amino acid position 448, this indicates the wild-typeIN301-303 polymorphism of SEQ ID NO: 7, since the mutant SEQ ID NO: 8does not have this amino acid position with regards to the encoded geneproduct. Thus, the IN/DEL301-303 can be indirectly detected by detectingone or more amino acid positions downstream of IN/DEL301-303 polymorphicsite.

[0082] The present invention includes fragments of gene products.Preferred gene product fragments of the alpha-2-BAR include less thanthe entire amino acid sequence of SEQ ID NO: 7 or 8. The fragmentscomprise the polymorphic site or are associated with the polymorphicsite. Preferred gene product fragments comprise the IN301-303 orDEL301-303 polymorphic site. In order for an amino acid sequence to be afragment, it must be readily identifiable by molecular andpharmacological techniques discussed below, such as for example, ligandbinding.

[0083] The present invention includes homologs and fragments of thenucleic acids that encode alpha2-BAR. To be considered a homolog oractive fragment, the sequence of an amino acid or a nucleic acid mustsatisfy two requirements. In the present specification, the sequence ofa first nucleotide sequence (SEQ ID NO: 1 or 2) is considered homologousto that of a second nucleotide sequence if the first sequence is atleast about 30% identical, preferably at least about 50% identical, andmore preferably at least about 65% identical to the second nucleotidesequence. In the case of nucleotide sequences having high homology, thefirst sequence is at least about 75%, preferably at least about 85%, andmore preferably at least about 95% identical to the second nucleotidesequence.

[0084] The amino acid sequence of a first protein (SEQ ID NO: 7 or 8) isconsidered to be homologous to that of a second protein if the aminoacid sequence of the first protein shares at least about 20% amino acidsequence identity, preferably at least about 40% identity, and morepreferably at least about 60% identity, with the sequence of the secondprotein. In the case of proteins having high homology, the amino acidsequence of the first protein shares at least about 75% sequenceidentity, preferably at least about 85% identity, and more preferably atleast about 95% identity, with the amino acid sequence of the secondprotein.

[0085] In order to compare a first amino acid or nucleic acid sequenceto a second amino acid or nucleic acid sequence for the purpose ofdetermining homology, the sequences are aligned so as to maximize thenumber of identical amino acid residues or nucleotides. The sequences ofhighly homologous proteins and nucleic acid molecules can usually bealigned by visual inspection. If visual inspection is insufficient,other methods of determining homology are known in the art. For example,the proteins may be aligned in accordance with the FASTA method inaccordance with Pearson et al. (1988). Preferably, any of the methodsdescribed by George et al., in Macromolecular Sequencing and Synthesis,Selected Methods and Applications, (1988).

[0086] A second test for homology of two nucleic acid sequences iswhether they hybridize under normal hybridization conditions, preferablyunder stringent hybridization conditions. Also included in the inventionare proteins that are encoded by nucleic acid molecules that hybridizeunder high stringent conditions to a sequence complementary to SEQ IDNO: 1 or 2. The term “stringent conditions,” as used herein, isequivalent to “high stringent conditions” and “high stringency”. Theseterms are used interchangeably in the art. High stringent conditions aredefined in a number of ways. In one definition, stringent conditions areselected to be about 50° C. lower than the thermal melting point (Tm)for a specific sequence at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched sequence. Typicalstringent conditions are those in which the salt concentration is atleast about 0.02 M at pH 7 and the temperature is at least about 60° C.“Stringent conditions,” in referring to homology or substantialsimilarity in the hybridization context, can be combined conditions ofsalt, temperature, organic solvents or other parameters that aretypically known to control hybridization reactions. The combination ofparameters is more important than the measure of any single parameter.If incompletely complementary sequences recognize each other under highstringency conditions, then these sequences hybridize under conditionsof high stringency. See U.S. Pat No. 5,786,210; Wetmur and Davidson J.Mol. Biol. 31:349-370 (1968). Control of hybridization conditions, andthe relationships between hybridization conditions and degree ofhomology are understood by those skilled in the art. See, e.g.,Sambrook, J. et al. (Eds.), Molecular Cloning, Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) andAusubel, F. M. et al. (Eds.), Current Protocols in Molecular Biology,John Wiley & Sons, New York (1999).

[0087] Substitutions, additions, and/or deletions in an amino acidsequence can be made as long as the protein encoded by the nucleic acidof the invention continues to satisfy the functional criteria describedherein. An amino acid sequence that is substantially the same as anothersequence, but that differs from the other sequence by means of one ormore substitutions, additions, and/or deletions, is considered to be anequivalent sequence. Preferably, less than 50%, more preferably lessthan 25%, and still more preferably less than 10%, of the number ofamino acid residues in a sequence are substituted for, added to, ordeleted from the protein encoded by the nucleic acid of the invention.

[0088] Alpha-2AAR Polymorphisms

[0089] The wild-type gene encoding the third intracellular loop of thehuman alpha-2A receptor molecule is disclosed in GenBank Accession No.AF281308 which include the sequence corrections illuminated by (13),both references are herein incorporated by reference. The terms“alpha-2A-adrenergic receptor polymorphism” or “alpha-2AARpolymorphism”, are terms of art and refer to at least one polymorphismin the nucleic acid or amino acid sequence of an alpha-2A adrenergicreceptor gene or gene product.

[0090] For purposes of the present application, the wild-type geneencoding the alpha-2A-adrenergic receptor is designated SEQ ID NO: 24and the wild-type gene product comprising the alpha-2A-adrenergicreceptor molecule, is designated amino acid SEQ ID NO: 26.

[0091] Preferred polymorphisms of the present invention occur in thegene encoding for the alpha-2A adrenergic receptor molecule identifiedas SEQ ID NO: 24 or 25 or fragments thereof or complements thereof.

[0092] For the purposes of identifying the location of at least onepolymorphism or polymorphic site, the first nucleotide of the startcodon of the coding region (the adenine of the ATG in a DNA molecule andthe adenine of the AUG in an RNA molecule) of the alpha-2AAR gene isconsidered nucleotide “1.” This corresponds to nucleotide 1 of SEQ IDNO: 24 or 25. The end of the coding region corresponds to guanine atposition 1350 of SEQ ID NO: 24 or 25. According to the presentinvention, polymorphisms can occur any where in the coding regionidentified as SEQ ID NO: 24 or 25.

[0093] Preferred single nucleotide polymorphisms and polymorphic sitesoccurring in the alpha-2AAR gene and the encoded protein or gene productinclude the following: TABLE 2 Preferred Alpha-2AAR PolymorphismsNucleotide Type Position Nucleot. Amino Acid position Designation Wild753 of SEQ C 251 of SEQ ID NO: 26 Asn 251 Type ID NO: 24 Mutant 753 ofSEQ G 251 of SEQ ID NO: 27 Lys 251 ID NO: 25

[0094] In one embodiment of the present invention, Applicants havediscovered at least one polymorphic site on SEQ ID NO: 24 that encodethe wild-type alpha-2AAR molecule (Table 2). Such polyimorphic sitecorresponds to C at nucleotide position 753 of SEQ ID NO: 24 in thecoding region of the alpha-2AAR molecule (FIG. 5B). This SNP islocalized within an intracellular domain of the alpha-2AAR molecule.

[0095] In another embodiment of the present invention at least onepolymorphic site has been identified in SEQ ID NO: 25 that encodes themutant alpha-2AAR molecule (Table 2). Such polymorphic site correspondsto G at nucleotide position 753 of SEQ ID NO: 25 in the coding regionfor the alpha-2AAR molecule (FIG. 5A). This SNP is localized within anintracellular domain of the alpha-2AAR molecule.

[0096] The polymorphisms of the present invention can occur in thetranslated alpha-2A adrenergic receptor molecule as well. For example,the first amino acid of the translated protein product or gene product(the methionine) is considered amino acid “1” in the wild-type or mutantalpha-2A adrenergic receptor molecule designated amino acid SEQ ID NO:26 or 27, respectively. Polymorphisms can occur anywhere in SEQ ID NO:26 or 27. The wild-type alpha-2A adrenergic receptor molecule (FIG. 6)comprises N at amino acid position 251 (Asn 251) of the alpha-2Aadrenergic receptor molecule. Accordingly, amino acid position 251 is apolymorphic site (Table 2).

[0097] In another embodiment of the present invention, SEQ ID NO: 27 isthe entire mutant amino acid sequence of alpha-2A adrenergic receptormolecule. Polymorphisms can occur anywhere in the amino acid sequencedesignated SEQ ID NO: 27. For example, the mutant alpha-2A adrenergicreceptor molecule comprises G at amino acid position 251 (Lys 251) ofthe alpha-2A adrenergic receptor. Accordingly, amino acid position 251is a polymorphic site (Table 2).

[0098] As used herein “fragments of the polynucleotide encoding thealpha-2A adrenergic receptor” include less than the entire nucleotidesequence of SEQ ID NO: 24 or 25. The fragments comprise the polymorphicsite or are associated with the polymorphic site. In order for a nucleicacid sequence to be a fragment, it must be readily identifiable by themolecular techniques as discussed below, such as with nucleic acidprobes. Preferred gene product fragments of the alpha-2A adrenergicreceptor include less than the entire amino acid sequence of SEQ ID NO:26 or 27. The fragments comprise the polymorphic site or are associatedwith the polymorphic site. In order for an amino acid sequence to be afragment, it must be readily identifiable by molecular andpharmacological techniques as discussed below, such as with ligandbinding.

[0099] Alpha-2CAR

[0100] The wild-type gene encoding the third intracellular loop of thehuman alpha-2C receptor molecule is disclosed in GenBank Accession No.AF280399, which is herein incorporated by reference. The terms“alpha-2C-adrenergic receptor polymorphism” or “alpha-2CARpolymorphism”, are terms of art and refer to at least one polymorphismin the nucleic acid or amino acid sequence of an alpha-2C adrenergicreceptor gene or gene product. For purposes of the present application,the wild-type gene encoding the alpha-2C-adrenergic receptor isdesignated SEQ ID NO: 40 and the wild-type gene product comprising thealpha-2C-adrenergic receptor molecule, is designated amino acid SEQ IDNO: 44. For the purposes of identifying the location of at least onepolymorphism or polymorphic site, the first nucleotide of the startcodon of the coding region (the adenine of the ATG in a DNA molecule andthe adenine of the AUG in an RNA molecule) of the alpha-2CAR gene isconsidered nucleotide “1.” This corresponds to nucleotide 1 of SEQ IDNO: 40. The end of the coding region corresponds to guanine at position1383 of SEQ ID NO: 40. According to the present invention, polymorphismscan occur any where in the coding region identified as SEQ ID NO: 40.TABLE 3 Preferred Alpha-2CAR Polymorphisms Amino Nucleotide Acid TypePosition Nucleotide position Designation Wild 964-975 ggggcggggccg322-325 IN322-325 GAGP Type of SEQ ID SEQ ID NO: 41 of SEQ ID SEQ ID NO:45 NO: 40 NO: 44 Mu- 964-975 ggggcggctgag 322-325 DEL322-325 GAAE tantof SEQ ID SEQ ID NO: 43 of SEQ ID SEQ ID NO: 47 NO: 42 NO: 46

[0101] For example, Applicants have discovered at least one polymorphicsite has been identified in SEQ ID NO: 40 corresponding to an insertionof nucleotides at positions 964-975 of SEQ ID NO: 40 which is the codingregion for the alpha-2CAR molecule (FIG. 11A). This polymorphism islocalized within an intracellular domain of the alpha-2CAR molecule. Thetwelve nucleotide insertion is listed as SEQ ID NO: 41 ggggcggggccgwhich is an example of a fragment or polymorphic site of SEQ ID NO: 40.

[0102] In another embodiment of the present invention at least onepolymorphic site has been identified in SEQ ID NO: 42. Such polymorphicsite is ggggcggctgag SEQ ID NO: 43. For example, the entire mutantnucleotide sequence encoding the alpha-2C adrenergic receptor moleculeis identified as SEQ ID NO: 42. Polymorphisms can occur in the codingregion identified as SEQ ID NO: 42. This polymorphic nucleotide sequencecomprises ggggcggctgag SEQ ID NO: 43 at positions 964-975. Thus, SEQ IDNO: 42 comprises a twelve nucleotide deletion (FIG. 11B) at nucleotidepositions 964-975 when compared to the wild-type acid sequenceidentified as SEQ ID NO: 40.

[0103] The polymorphisms of the present invention can occur in thetranslated alpha-2C adrenergic receptor molecule as well. For example,the first amino acid of the translated protein product or gene product(the methionine) is considered amino acid “1” in the wild-type alpha-2Cadrenergic receptor molecule designated amino acid SEQ ID NO: 44.Polymorphisms can occur anywhere in SEQ ID NO: 44. The wild-typealpha-2C adrenergic receptor molecule (FIG. 12) comprises GAGP at aminoacid positions 322-325 of the alpha-2C adrenergic receptor moleculedesignated amino acid SEQ ID NO: 45. GAGP at amino acid positions322-325 of the alpha-2C adrenergic receptor molecule is an example of afragment or polymorphic site of SEQ ID NO: 44.

[0104] In another embodiment of the present invention, SEQ ID NO: 46 isthe entire mutant amino acid sequence of alpha-2C adrenergic receptormolecule with deletion of GAGP at amino acid positions 322-325 (shown inFIG. 12). Polymorphisms can occur anywhere in the amino acid sequencedesignated SEQ ID NO: 46. For example, the mutant alpha-2C adrenergicreceptor molecule comprises GAAE at amino acid positions 322-325 inalpha-2C adrenergic receptor molecule designated amino acid SEQ ID NO:47. GAAE at amino acid positions 322-325 alpha-2C adrenergic receptormolecule is an example of a fragment or polymorphic site of SEQ ID NO:46. As used herein “fragments of the polynucleotide encoding thealpha-2C adrenergic receptor” include less than the entire nucleotidesequence of SEQ ID NO: 40 or 42. The fragments comprise the polymorphicsite or are associated with the polymorphic site. In order for a nucleicacid sequence to be a fragment, it must be readily identifiable by themolecular techniques as discussed below, such as with nucleic acidprobes. Preferred gene product fragments of the alpha-2C adrenergicreceptor include less than the entire amino acid sequence of SEQ ID NO:44 or 46. The fragments comprise the polymorphic site or are associatedwith the polymorphic site. In order for an amino acid sequence to be afragment, it must be readily identifiable by molecular andpharmacological techniques as discussed below, such as with ligandbinding.

[0105] In another embodiment, the present invention provides a computerreadable medium recorded thereon the nucleotide sequence of SEQ ID NO:43. As used herein, “computer readable medium” includes files in any ofthe following media: hard drive, diskette, magnetic tape, 8mm datacartridge, compact disk and Magneto Optical Disk, and the like.

[0106] Alpha-2B, Alpha-2A, Alpha-2C Adrenergic Receptor Molecules

[0107] The molecules of the present invention are particularly relevantto determine increased risk an individual has for a disease and/orresponse to therapy. The molecules of the present invention can also beused to diagnosis and prognosis a disease.

[0108] The molecules of the present invention will preferably be“biologically active” with respect to either a structural attribute,such as the capacity of a nucleic acid to hybridize to another nucleicacid molecule or to be used by a polymerase as a primer. Alternatively,such an attribute may be catalytic, and thus involve the capacity of theagent to mediate a chemical reaction or response.

[0109] A preferred class of molecules of the present invention comprisesadrenergic receptor molecules. Preferably, alpha-2B, alpha-2A, oralpha-2C adrenergic receptor molecules. These molecules may be eitherDNA or RNA, single-stranded or double-stranded. Alternatively, suchmolecules may be proteins and antibodies. These molecules may also befragments, portions, and segments thereof and molecules, such asoligonucleotides, that specifically hybridize to nucleic acid moleculesencoding the alpha-2B, alpha-2A, or alpha-2C adrenergic receptor. Suchmolecules may be isolated, derived, or amplified from a biologicalsample. Alternatively, the molecules of the present invention may bechemically synthesized. The term “isolated” as used herein refers to thestate of being substantially free of other material such as nucleicacids, proteins, lipids, carbohydrates, or other materials such ascellular debris or growth media with which the alpha-2B, alpha-2A, oralpha-2C adrenergic receptor molecule, polynucleotide encoding thealpha-2B, alpha-2A, or alpha-2C adrenergic receptor molecule, primeroligonucleotide, or allele-specific oligonucleotide may be associated.Typically, the term “isolated” is not intended to refer to a completeabsence of these materials. Neither is the term “isolated” generallyintended to refer to water, buffers, or salts, unless they are presentin amounts that substantially interfere with the methods of the presentinvention. The term “sample” as used herein generally refers to anymaterial containing nucleic acid, either DNA or RNA or amino acids.Generally, such material will be in the form of a blood sample, stoolsample, tissue sample, cells, bacteria, histology section, or buccalswab, either fresh, fixed, frozen, or embedded in paraffin.

[0110] As used herein, the term “polynucleotide” includes nucleotides ofany number. A polynucleotide includes a nucleic acid molecule of anynumber of nucleotides including single-stranded RNA, DNA or complementsthereof, double-stranded DNA or RNA, and the like. Preferredpolynucleotides include SEQ ID NO: 1, 2, 24, 25, 40 or 42 andcomplements and fragments thereof. The term “oligonucleotide” as usedherein includes a polynucleotide molecule comprising any number ofnucleotides, preferably, less than about 200 nucleotides. Morepreferably, oligonucleotides are between 5 and 100 nucleotides inlength. Most preferably, oligonucleotides are 10 to 50 nucleotides inlength. The exact length of a particular oligonucleotide, however, willdepend on many factors, which in turn depend on the ultimate function oruse of the oligonucleotide. Short primer molecules generally requirelower temperatures to form sufficiently stable hybrid complexes with thetemplate. Preferred oligonucleotides associated with the alpha-2BARinclude: 5′-GCTCATCATCCCTTTCTCGCT-3′; (SEQ ID NO:13)5′-AAAGCCCCACCATGGTCGGGT-3′; (SEQ ID NO:14) 5′-CTGATCGCCAAACGAGCAAC-3′;(SEQ ID NO:15) 5′-AAAAACGCCAATGACCACAG-3′ (SEQ ID NO:16)5′-TGTAAAACGACGGCCAGT-3′; (SEQ ID NO:17) and 5′-CAGGAAACAGCTATGACC-3′;(SEQ ID NO:18) 5′-AGAAGGAGGGTGTTTGTGGGG-3′; (SEQ ID NO:19)5′-ACCTATAGCACCCACGCCCCT-3′; (SEQ ID NO:20) 5′-GGCCGACGCTCTTGTCTAGCC-3′;(SEQ ID NO:21) 5′-CAAGGGGTTCCTAAGATGAG-3′; (SEQ ID NO:22) andcomplementary sequences thereof.

[0111] Preferred oligonucleotides associated with the alpha-2AARinclude: 5′-TTTACCCATCGGCTCTCCCTAC-3′; (SEQ ID NO:28)5′GAGACACCAGGAAGAGGTTTTGG-3′; (SEQ ID NO:29) 5′-TCGTCATCATCGCCGTGTTC-3′;(SEQ ID NO:30) 5′-CGTACCACTTCTGGTCGTTGATC-3′; (SEQ ID NO:31)5′-GCCATCATCATCACCGTGTGGGTC-3′; (SEQ ID NO:32)5′-GGCTCGCTCGGGCCTTGCCTTTG-3′; (SEQ ID NO:33)5′-GACCTGGAGGAGAGCTCGTCTT-3′; (SEQ ID NO:34)5′-TGACCGGGTTCAACGAGCTGTTG-3′; (SEQ ID NO:35)5′-GCCACGCACGCTCTTCAAATTCT-3′; (SEQ ID NO:36)5′-TTCCCTTGTAGGAGCAGCAGAC-3′; (SEQ ID NO:37) 5′-TGTAAAACGACGGCCAGT-3′;(SEQ ID NO:38) 5′-CAGGAAACAGCTATGACC-3′ (SEQ ID NO:39) and complementarysequences thereof.

[0112] Preferred oligonucleotides associated with the alpha-2CARinclude: 5′-CCACCATCGTCGCCGTGTGGCTCATCT-3′, (SEQ ID NO:48)5′-AGGCCTCGCGGCAGATGCCGTACA-3′, (SEQ ID NO:49)5′-AGCCGGACGAGAGCAGCGCA-3′, (SEQ ID NO:50) and complementary sequencesthereof.

[0113] Oligonucleotides, such as primer oligonucleotides are preferablysingle stranded, but may alternatively be double stranded. If doublestranded, the oligonucleotide is generally first treated to separate itsstrands before being used for hybridization purposes or being used toprepare extension products. Preferably, the oligonucleotide is anoligodeoxyribonucleotide. Oligonucleotides may be synthesized chemicallyby any suitable means known in the art or derived from a biologicalsample, as for example, by restriction digestion. The source of theoligonucleotides is not essential to the present invention.Oligonucleotides may be labeled, according to any technique known in theart, such as with radiolabels, fluorescent labels, enzymatic labels,proteins, haptens, antibodies, sequence tags, mass tags, fluorescentpolarization etc. The term “nucleotide” or nucleic acid as used hereinis intended to refer to ribonucleotides, deoxyribonucleotides, acylicderivatives of nucleotides, and functional equivalents thereof, of anyphosphorylation state. Functional equivalents of nucleotides are thosethat act as substrates for a polymerase as, for example, in anamplification method. Functional equivalents of nucleotides are alsothose that may be formed into a polynucleotide that retains the abilityto hybridize in a sequence specific manner to a target polynucleotide.

[0114] Such oligonucleotides may be used as probes of a nucleic acidsample, such as genomic DNA, mRNA, or other suitable sources of nucleicacid. For such purposes, the oligonucleotides must be capable ofspecifically hybridizing to a target polynucleotide or DNA nucleic acidmolecule. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure under hybridizing conditions, whereas they are substantiallyunable to form a double-stranded structure when incubated with anon-alpha-2BAR, a non-alpha-2AAR or a non-alpha-2CAR nucleic acidmolecule under the same conditions. A nucleic acid molecule is said tobe the “complement” of another nucleic acid molecule if it exhibitscomplete complementarity. As used herein, molecules are said to exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are said tobe “substantially complementary” if they can hybridize to one anotherwith sufficient stability to permit them to remain annealed to oneanother under at least conventional “low-stringency” conditions.Similarly, the molecules are said to be “complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under conventional “high-stringency”conditions. Conventional stringency conditions are described, forexample, by Sambrook, J., et al., in Molecular Cloning, a LaboratoryManual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989), and by Haymes, B. D., et al. in Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985), both hereinincorporated by reference). Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure.

[0115] For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being complementary to the strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the strand to hybridize therewith for the purposesemployed. However, for detection purposes, particularly using labeledsequence-specific probes, the primers typically have exactcomplementarity to obtain the best results. Thus, for an oligonucleotideto serve as an allele-specific oligonucleotide, it must generally becomplementary in sequence and be able to form a stable double-strandedstructure with a target polynucleotide under the particularenvironmental conditions employed.

[0116] The term “allele-specific oligonucleotide” refers to anoligonucleotide that is able to hybridize to a region of a targetpolynucleotide spanning the sequence, mutation, or polymorphism beingdetected and is substantially unable to hybridize to a correspondingregion of a target polynucleotide that either does not contain thesequence, mutation, or polymorphism being detected or contains analtered sequence, mutation, or polymorphism. As will be appreciated bythose in the art, allele-specific is not meant to denote an absolutecondition. Allele-specificity will depend upon a variety ofenvironmental conditions, including salt and formamide concentrations,hybridization and washing conditions and stringency. Depending on thesequences being analyzed, one or more allele-specific oligonucleotidesmay be employed for each target polynucleotide. Preferably,allele-specific oligonucleotides will be completely complementary to thetarget polynucleotide. However, departures from complete complementarityare permissible. In order for an oligonucleotide to serve as a primeroligonucleotide, however, it typically need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular environmental conditions employed.Establishing environmental conditions typically involves selection ofsolvent and salt concentration, incubation temperatures, and incubationtimes. The terms “primer” or “primer oligonucleotide” as used hereinrefer to an oligonucleotide as defined herein, which is capable ofacting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension product which iscomplementary to a nucleic acid strand is induced, as for example, in aPCR reaction. As with non-primer oligonucleotides, primeroligonucleotides may be labeled, according to any technique known in theart, such as with radiolabels, fluorescent labels, enzymatic labels,proteins, haptens, antibodies, sequence tags, and the like.

[0117] In performing the methods of the present invention, theoligonucleotides or the target polynucleotide may be either in solutionor affixed to a solid support. Generally, allele-specificoligonucleotides will be attached to a solid support, though in certainembodiments of the present invention allele-specific oligonucleotidesmay be in solution. In some such embodiments, the target polynucleotideis preferably bound to a solid support. In those embodiments where theallele-specific oligonucleotides or the target polynucleotides areattached to a solid support, attachment may be either covalent ornon-covalent. Attachment may be mediated, for example, byantibody-antigen-type interactions, poly-L-Lys, streptavidin oravidin-biotin, salt-bridges, hydrophobic interactions, chemicallinkages, LTV cross-ag, baking, and the like. In addition,allele-specific oligonucleotides can be synthesized directly on a solidsupport or attached to the solid support subsequent to synthesis. In apreferred embodiment, allele-specific oligonucleotides are affixed on asolid support such that a free 3′-OH is available forpolymerase-mediated primer extension.

[0118] Suitable solid supports for the present invention includesubstrates constructed of silicon, glass, plastic (polystyrene, nylon,polypropylene, etc.), paper, etc. Solid supports may be formed, forexample, into wells (as in 96-well dishes), plates, slides, sheets,membranes, fibers, chips, dishes, and beads. The solid support can be anarray of nucleotides with different discrete nucleotide sequences atpositions on the arrays. In certain embodiments of the presentinvention, the solid support is treated, coated, or derivatized so as tofacilitate the immobilization of an allele-specific oligonucleotide or atarget polynucleotide. Preferred treatments include coating, treating,or derivatizing with poly-L-Lys, streptavidin, antibodies, silanederivatives, low salt, or acid.

[0119] Providing Alpha-2B, Alpha-2A and Alpha-2C Adrenergic ReceptorMolecules

[0120] The nucleic acid molecules or DNA encoding the alpha-2B, alpha-2Aor alpha-2C adrenergic receptor molecule identified as SEQ ID NO: 1 or2, SEQ ID NO: 24 or 25, or SEQ ID NO: 40 or 42, respectively, of theinvention may be obtained from a sample. The alpha-2B, alpha-2A, andalpha-2C-adrenergic receptor molecules (amino acids) can be obtainedfrom the sample as well.

[0121] DNA encoding the protein (alpha-2B, alpha-2A, andalpha-2C-adrenergic receptor molecules) may also be chemicallysynthesized by methods known in the art. Suitable methods forsynthesizing the protein are described by Stuart and Young in SolidPhase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984),Solid Phase Peptide Synthesis, Methods Enzymol., 289, Academic Press,Inc, New York (1997). Suitable methods for synthesizing DNA aredescribed by Caruthers in Science 230:281-285 (1985) and “DNA Structure,Part A: Synthesis and Physical Analysis of DNA,” Lilley, D. M. J. andDahlberg, J. E. (Eds.), Methods Enzymol., 211, Academic Press, Inc., NewYork (1992).

[0122] DNA may also be synthesized by preparing overlappingdouble-stranded oligonucleotides, using PCR, filling in the gaps, andligating the ends together. The DNA may be cloned in a suitablerecombinant host cell and expressed. The DNA and protein may berecovered from the host cell. See, generally, Sambrook, J. et al.(Eds.), Molecular Cloning, Second Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989) and Ausubel, F. M. et al. (Eds.),Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork (1999).

[0123] The nucleic acid molecules or DNA encoding the alpha-2B, alpha-2Aor alpha-2C adrenergic receptor molecule identified as SEQ ID NO: 1 or2, SEQ ID NO: 24 or 25, or SEQ ID NO: 40 or 42, respectively, of theinvention may be replicated and used to express recombinant proteinfollowing insertion into a wide variety of host cells in a wide varietyof cloning and expression vectors. The host may be prokaryotic oreukaryotic. The DNA may be obtained from natural sources and,optionally, modified. The genes may also be synthesized in whole or inpart.

[0124] Cloning vectors may comprise segments of chromosomal,non-chromosomal and synthetic DNA sequences. Some suitable prokaryoticcloning vectors include plasmids from E. coli, such as colE1, pCR1,pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also includederivatives of phage DNA such as M13 and other filamentoussingle-stranded DNA phages.

[0125] Vectors for expressing proteins in bacteria, especially E. coli,are also known. Such vectors include the pK233 (or any of the tac familyof plasmids), T7, pBluescript II, bacteriophage lamba ZAP, and lambdaP_(L) (Wu, R. (Ed.), Recombinant DNA Methodology II, Methods Enzymol.,Academic Press, Inc., New York, (1995)). Examples of vectors thatexpress fusion proteins are PATH vectors described by Dieckmann andTzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors containDNA sequences that encode anthranilate synthetase (TrpE) followed by apolylinker at the carboxy terminus. Other expression vector systems arebased on beta-galactosidase (pEX); maltose binding protein (pMAL);glutathione S-transferase (pGST or PGEX)—see Smith, D. B. Methods Mol.Cell Biol. 4:220-229 (1993); Smith, D. B. and Johnson, K. S., Gene67:31-40 (1988); and Peptide Res. 3:167 (1990), and TRX (thioredoxin)fusion protein (TRXFUS)—see LaVallie, R. et al., Bio/Technology11:187-193 (1993). A particularly preferred plasmid of the presentinvention is pBC12BI.

[0126] Vectors useful for cloning and expression in yeast are available.Suitable examples are 2 μm circle plasmid, Ycp50, Yep24, Yrp7, Yip5, andpYAC3 (Ausubel, F. M. et al. (Eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc., New York, (1999)).

[0127] Suitable cloning/expression vectors for use in mammalian cellsare also known. Such vectors include well-known derivatives of SV-40,adenovirus, cytomegalovirus (CMV) retrovirus-derived DNA sequences. Anysuch vectors, when coupled with vectors derived from a combination ofplasmids and phage DNA, i.e. shuttle vectors, allow for the isolationand identification of protein coding sequences in prokaryotes.

[0128] Further eukaryotic expression vectors are known in the art (e.g.,P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982); S.Subramani et al., Mol. Cell. Biol. 1:854-864 (1981); R. J. Kaufmann andP. A. Sharp, “Amplification And Expression Of Sequences Cotransfectedwith A Modular Dihydrofolate Reductase Complementary DNA Gene,” J. Mol.Biol. 159:601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell.Biol. 159:601-664 (1982); S. I. Scahill et al, “Expression AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells,” Proc. Natl. Acad. Sci. USA 80:4654-4659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA77:4216-4220 (1980).

[0129] The expression vectors useful in the present invention contain atleast one expression control sequence that is operatively linked to theDNA sequence or fragment to be expressed. The control sequence isinserted in the vector in order to control and to regulate theexpression of the cloned DNA sequence. Examples of useful expressioncontrol sequences are the lac system, the trp system, the tac system,the trc system, the tet system, major operator and promoter regions ofphage lambda, the control region of fd coat protein, the glycolyticpromoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase,the promoters of yeast acid phosphatase, e.g., Pho5, the promoters ofthe yeast alpha-mating factors, and promoters derived from polyoma,adenovirus, retrovirus, and simian virus, e.g., the early and latepromoters or SV40, and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells and their viruses orcombinations thereof.

[0130] Useful expression hosts include well-known prokaryotic andeukaryotic cells. Some suitable prokaryotic hosts include, for example,E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coliX1776, E. coli X2282, E. coli DH1, E. coli DH5alphaF, and E. coli MRCl,Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.Suitable eukaryotic cells include yeasts and other fungi, insect, animalcells, such as COS cells and CHO cells, human cells and plant cells intissue culture. A particularly preferred host cell is CHO.

[0131] The alpha-2B adrenergic receptor molecule identified as SEQ IDNO: 7 or 8 can be the entire protein as it exists in nature isolatedfrom the sample, or an antigenic, preferably immunogenic, fragment ofthe whole protein. The alpha-2A adrenergic receptor molecule identifiedas SEQ ID NO: 26 or 27 can be the entire protein as it exists in nature,or an antigenic, preferably immunogenic, fragment of the whole protein.The alpha-2C adrenergic receptor molecule identified as SEQ ID NO: 44 or46 may be the entire protein as it exists in nature, or an antigenic,preferably immunogenic, fragment of the whole protein. Antigenic and/orimmunogenic fragments of antigenic and/or immunogenic proteins may beidentified by methods known in the art.

[0132] Fragments containing antigenic sequences may be selected on thebasis of generally accepted criteria of potential antigenicity and/orexposure. Such criteria include the hydrophilicity and relativeantigenic index, as determined by surface exposure analysis of proteins.The determination of appropriate criteria is known to those skilled inthe art, and has been described, for example, by Hopp, T., MethodsEnzymol., 178:571-585 Academic Press, Inc., New York (1989); Becker, Y.,Virus Genes 6:79-93 (1992); Regenmortel, V and Pellequer, J. L., Pept.Res. 7:224-228 (1994); Gallet, X. et al., Prot. Eng. 8:829-834 (1995);Kyte et al., J. Mol. Biol. 157:105-132 (1982); Emini, E. A. et al., J.Virol. 55:836-839 (1985); Jameson et al., CA BIOS 4:181-186 (1988); andKarplus et al., Naturwissenschaften 72:212-213 (1985). Amino aciddomains predicted by these criteria to be surface exposed are selectedpreferentially over domains predicted to be more hydrophobic or hidden.

[0133] Methods for isolating and identifying antigenic fragments fromknown antigenic proteins are described by Salfeld et al. in J. Virol.63:798-808 (1989) and by Isola et al. in J. Virol. 63:2325-2334 (1989).An alternative means for identifying antigenic sites on protein is bythe use of synthetic peptide combinatorial library or phage-displaypeptide library as described in Combinatorial Peptide Library Protocols,Cabilly, S. (Ed.), Humana Press, New York, 1998; Pinilla, C. et al.,Pept. Res. 8:250-257 (1995); Scala, G. et al., J. Immunol. 162:6155-6161(1999); Pereboeva, L. A. et al., J. Med. Virol. 56:105-111 (1998); andDemkowicz, W. E. et al., J. Virol. 66:386-398 (1992).

[0134] The alpha-2B, alpha-2A and alpha-2C adrenergic receptor moleculesare isolated from the sample by standard methods known in the art. Somesuitable methods include precipitation and liquid/chromatographicprotocols such as ion exchange, hydrophobic interaction and gelfiltration See, for example, Guide to Protein Purification, Deutscher,M. P. (Ed.) Methods Enzymol., 182, Academic Press, Inc., New York (1990)and Scopes, R. K. and Cantor, C. R. (Eds.), Protein Purification (3d),Springer-Verlag, N.Y. (1994).

[0135] Alternatively, purified material is obtained by separating theprotein on preparative SDS-PAGE gels, slicing out the band of interestand electroeluting the protein from the polyacrylamide matrix by methodsknown in the art. The detergent SDS is removed from the protein by knownmethods, such as by dialysis or the use of a suitable column, such asthe Extracti-Gel column from Pierce.

[0136] Detection of Polymorphisms

[0137] The polymorphisms of the present invention may be detecteddirectly or indirectly using any of a variety of suitable methods.Indirect methods include detecting the nucleotides on the complementarystrand of DNA or detecting nucleotide shifts upstream or downstream ofthe polymorphic site. One method of detection of nucleotides is byfluorescent techniques. Fluorescent hybridization probes may beconstructed that are quenched in the absence of hybridization to targetnucleic acid sequences. Other methods capitalize on energy transfereffects between fluorophores with overlapping absorption and emissionspectra, such that signals are detected when two fluorophores are inclose proximity to one another, as when captured or hybridized.

[0138] Nucleotides may also be detected by, or labeled with moietiesthat can be detected by, a variety of spectroscopic methods relating tothe behavior of electromagnetic radiation. These spectroscopic methodsinclude, for example, electron spin resonance, optical activity orrotation spectroscopy such as circular dichroism spectroscopy,fluorescence polarization, absorption/emission spectroscopy,ultraviolet, infrared, or mass spectroscopy, Raman spectroscopy, visiblespectroscopy, and nuclear magnetic resonance spectroscopy.

[0139] The term “detection” refers to identification of a detectablemoiety or moieties. The term is intended to include the ability toidentify a moiety by electromagnetic characteristics, such as, forexample, charge, light, fluorescence, chemiluminescense, changes inelectromagnetic characteristics such as, for example, fluorescencepolarization, light polarization, dichroism, light scattering, changesin refractive index, reflection, infrared, ultraviolet, and visiblespectra, and all manner of detection technologies dependent uponelectromagnetic radiation or changes in electromagnetic radiation. Theterm is also intended to include identification of a moiety based onbinding affinity, intrinsic mass, mass deposition, and electrostaticproperties.

[0140] Single channel detection refers to instrumentation or methodslimited to simultaneous or non-simultaneous detection of a singlecharacteristic of a detectable moiety or moieties. Bi-channel detectionrefers to instrumentation or methods of simultaneous or non-simultaneousdetection of a characteristic of a detectable moiety or moieties.Multiple-channel detection refers to instrumentation or methods limitedto simultaneous or non-simultaneous detection of or more characteristicof a detectable moiety or moieties.

[0141] One single channel platforms suitable for use with the presentinvention is the Luminex LabMAP system which is limited to a singleassay result readout channel, having only a single laser and a singlephotomultiplier to image assay output. In the LabMAP platform, a singlebiotinylated nucleotide is labeled after a reaction run with afluorophore for detection by flow cytometry.

[0142] Another single channel detection system suitable for use with thepresent invention is the BioStar platform. This detection system employsa unique thin-film preparation of a silicon surface that can be reactedfor highly sensitive assay readout. As currently available, a singleELISA step with a precipitating is used to generate a signal. Signaldetection, however, is achieved through a change of mass on the surfacerather than by color per se, although a color change is a simple methodof imaging the mass change.

[0143] Another method of detecting the nucleotide present at thepolymorphic site is by comparison of the concentrations of free,unincorporated nucleotides remaining in the reaction mixture at anypoint after the primer extension reaction. Mass spectroscopy in generaland, for example, electrospray mass spectroscopy, may be employed forthe detection of unincorporated nucleotides in this embodiment. Thisdetection method is possible because only the nucleotide(s)complementary to the polymorphic base is (are) depleted in the reactionmixture during the primer extension reaction. Thus, mass spectrometrymay be employed to compare the relative intensities of the mass peaksfor the nucleotides, Likewise, the concentrations of unlabeled primersmay be determined and the information employed to arrive at the identityof the nucleotide present at the polymorphic site.

[0144] One particularly preferred array is the GenFlex™ Tag Array, fromAffymetrix, Inc., that is comprised of capture probes for 2000 tagsequences. These are 20mers selected from all possible 20 mers to havesimilar hybridization characteristics and at least minimal homology tosequences in the public databases.

[0145] Another preferred array is the addressable array that has reversecomplements to the unique 5′ tags of the upper and lower primers. Thesereverse complements are bound to the array at known positions. This typeof tag hybridizes with the array under suitable hybridizationconditions. By locating the bound primer in conjunction with detectingone or more extended primers, the nucleotide identity at the polymorphicsite can be determined.

[0146] In one preferred embodiment of the present invention, the targetnucleic acid sequences are arranged in a format that allows multiplesimultaneous detections (multiplexing), as well as parallel processingusing oligonucleotide arrays.

[0147] In another embodiment, the present invention includes virtualarrays where extended and unextended primers are separated on an arraywhere the array comprises a suspension of microspheres, where themicrospheres bear one or more capture moieties to separate the uniquelytagged primers. The microspheres, in turn, bear unique identifyingcharacteristics such that they are capable of being separated on thebasis of that characteristic, such as for example, diameter, density,size, color, and the like.

[0148] Suitable methods comprise direct or indirect sequencing methods,restriction site analysis, hybridization methods, nucleic acidamplification methods, gel migration methods, the use of antibodies thatare specific for the proteins encoded by the different alleles of thepolymorphism, or by other suitable means. Alternatively, many suchmethods are well known in the art and are described, for example in T.Maniatis et al., Molecular Cloning, a Laboratory Manual, 2nd Edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), J. W. Zyskindet al., Recombinant DNA Laboratory Manual, Academic Press, Inc., NewYork (1988), and in R. Elles, Molecular Diagnosis of Genetic Diseases,Humana Press, Totowa, New Jersey (1996), each herein incorporated byreference.

[0149] Exemplary antibody molecules for detecting the alpha-2BAR,alpha-2AAR, or alpha-2CAR amino acid variants are intact immunoglobulinmolecules, substantially intact immunoglobulin molecules, or thoseportions of immunoglobulin molecules that contain the antigen bindingsite. Polyclonal or monoclonal antibodies may be produced by methodsconventionally known in the art (e.g., Kohler and Milstein, Nature,256:495-497 (1975); Campbell “Monoclonal Antibody Technology, theProduction and Characterization of Rodent and Human Hybridomas”, 1985,In: “Laboratory Techniques in Biochemistry and Molecular Biology,” Eds.Burdon et al., Volume 13, Elsevier Science Publishers, Amsterdam). Theantibodies or antigen binding fragments thereof may also be produced bygenetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is described in Huse et al., 1989, Science246:1275-1281. The antibodies may also be humanized (e.g., Queen, C. etal. 1989 Proc. Natl. Acad. Sci. 86;10029).

[0150] Identification methods may be of either a positive-type or anegative-type. Positive-type methods determine the identity of anucleotide contained in a polymorphic site, whereas negative-typemethods determine the identity of a nucleotide not present in apolymorphic site. Thus, a wild-type site may be identified either aswild-type or not mutant. For example, at a biallelic polymorphic sitewhere the wild-type allele contains a cytosine and the mutant allelecontains adenine, a site may be positively determined to be eitheradenine or cytosine or negatively determined to be not adenine (and thuscytosine) or not cytosine (and thus adenine).

[0151] Alternately, if the polymorphism is a deletion, or addition thenthe complementary sequence can be detected. As another example, inhybridization-based assay, a target polynucleotide containing a mutatedsite may be identified positively by hybridizing to an allele-specificoligonucleotide containing the mutated site or negatively, by failing tohybridize to a wild-type allele-specific oligonucleotide. Similarly, arestriction site may be determined to be present or lacking.

[0152] Direct Sequencing

[0153] Direct sequencing by methods such as dideoxynucleotide sequencing(Sanger), cycle sequencing, or Maxam-Gilbert sequencing are examples ofsuitable methods for determining the identity of a nucleotide at apolymorphic site of a target polynucleotide. Such methods are widelyknown in the art and are discussed at length, in the above-cited texts.

[0154] Both the dideoxy-mediated method and the Maxam-Gilbert method ofDNA sequencing require the prior isolation of the DNA molecule which isto be sequenced. The sequence information is obtained by subjecting thereaction products to electrophoretic analysis (typically usingpolyacrylamide gels). Thus, a sample is applied to a lane of a gel, andthe various species of nested fragments are separated from one anotherby their migration velocity through the gel. The number of nestedfragments which can be separated in a single lane is approximately200-300 regardless of whether the Sanger or the Maxam-Gilbert method isused. Thus, in order to identify a nucleotide at a particularpolymorphic site in a target polynucleotide, extraneous sequenceinformation is typically produced. The chief advantage of directsequencing lies in its utility for locating previously unidentifiedpolymorphic sites.

[0155] One of the problems that has encumbered the development of usefulassays for genetic polymorphisms is that in many cases, it is desirableto determine the identity of multiple polymorphic loci. This frequentlyrequires sequencing significant regions of the genome or performingmultiple assays with an individual patient sample.

[0156] Restriction Site Analysis

[0157] Restriction enzymes are specific for a particular nucleotidesequence. In certain embodiments of the present invention, the identityof a nucleotide at a polymorphic site is determined by the presence orabsence of a restriction enzyme site. A large number of restrictionenzymes are known in the art and, taken together, they are capable ofrecognizing at least one allele of many polymorphisms.

[0158] This feature of restriction enzymes may be utilized in a varietyof methods for identifying a polymorphic site. Restriction fragmentlength polymorphism (RFLP) analysis is an example of a suitable methodfor identifying a polymorphic site with restriction enzymes (Lentes etal., Nucleic Acids Res. 16:2359 (1988); and C. K. McQuitty et al., Hum.Genet. 93:225 (1994)). In RFLP analysis, at least one targetpolynucleotide is digested with at least one restriction enzyme and theresultant “restriction fragments” are separated based on mobility in agel. Typically, smaller fragments migrate faster than larger fragments.Consequently, a target polynucleotide that contains a particularrestriction enzyme recognition site will be digested into two or moresmaller fragments, which will migrate faster than a larger fragmentlacking the restriction enzyme site. Knowledge of the nucleotidesequence of the target polynucleotide, the nature of the polymorphicsite, and knowledge of restriction enzyme recognition sequences guidethe design of such assays.

[0159] Hybridization

[0160] Several suitable hybridization-based methods for identifying anucleotide at a polymorphic site have been described. Generally,allele-specific oligonucleotides are utilized in performing suchhybridization-based methods. Preferably, allele-specificoligonucleotides are chosen that are capable of specifically hybridizingto only one allele of an alpha-2B, an alpha-2A, or an alpha-2C moleculeat a region comprising a polymorphic site. In those embodiments whereinmore than one polymorphic site is identified, sets of allele-specificoligonucleotides are preferably chosen that have melting temperatureswithin 5° C. of each other when hybridizing to their completecomplement. Most preferably, such sets of allele-specificoligonucleotides are chosen so as to have melting temperatures within20° C. of each other. Examples of suitable hybridization methods aredescribed in standard manuals such as Molecular Cloning, A LaboratoryManual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor);and Current Protocols in Molecular Biology (Eds. Ausubel, Brent,Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc.,Wiley-Interscience, NY, N.Y., 1992) or that are otherwise known in theart. Examples of preferred hybridization methods include Southern,northern, and dot blot hybridizations, allele-specific oligonucleotidehybridizations (Hall et al., The Lancet 345:1213-1214 (1995)), reversedot blot hybridizations (Sakai et al., Nucl. Acids. Res. 86:6230-6234(1989)), DNA chip hybridizations (Drmanac et al., U.S. Pat. No.5,202,231), and hybridizations to allele-specific oligonucleotides.

[0161] Macevicz (U.S. Pat. No. 5,002,867), for example, describes amethod for deriving nucleic acid sequence information via hybridizationwith multiple mixtures of oligonucleotide probes. In accordance withsuch method, the sequence of a target polynucleotide is determined bypermitting the target to sequentially hybridize with sets of probeshaving an invariant nucleotide at one position, and variant nucleotidesat other positions. The Macevicz method determines the nucleotidesequence of the target by hybridizing the target with a set of probes,and then determining the number of sites that at least one member of theset is capable of hybridizing to the target (i.e. the number of“matches”). This procedure is repeated until each member of a sets ofprobes has been tested.

[0162] Polymerase-Mediated Primer Extension

[0163] The “Genetic Bit Analysis” (“GBA”) method disclosed by Goelet, P.et al. (WO92/15712, and U.S. Pat. Nos. 5,888,819 and 6,004,744, allherein incorporated by reference), is a preferred method for determiningthe identity of a nucleotide at a predetermined polymorphic site in atarget polynucleotide. The target polynucleotide can be, for example,nucleic acids encoding the alpha-2B, alpha-2C, or alpha-2A adrenergicreceptor molecule or complements or fragments thereof. GBA is a methodof polymorphic site interrogation in which the nucleotide sequenceinformation surrounding a polymorphic site in a target nucleic acidsequence is used to design an oligonucleotide primer that iscomplementary to a region immediately adjacent to at the 3′ or 5′ end ofthe target polynucleotide, but not including, the variable nucleotide(s)in the polymorphic site of the target polynucleotide. The targetpolynucleotide is isolated from the biological sample and hybridized tothe interrogating primer. In some embodiments of the present invention,following isolation, the target polynucleotide may amplified by anysuitable means prior to hybridization to the interrogating primer. Theprimer is extended by a single labeled terminator nucleotide, such as adideoxynucleotide, using a polymerase, often in the presence of one ormore chain terminating nucleoside triphosphate-precursors (or suitable,analogs). A detectable signal is thereby produced.

[0164] For example, to detect the polymorphic site on target nucleicacids encoding the alpha-2BAR, a primer oligonucleotide complementary toa region of SEQ I) NO: 1 can be hybridized at the primer's 3′ end to aregion up to and including, for example, nucleotide position number 904of SEQ ID NO: 1. If the primer is extended at its 3′ end by a singlelabeled terminator nucleotide, such as for example the dideoxynucleotideddTTP, using a polymerase, a detectable signal is produced indicatingthe complementary nucleotide A at nucleotide position 903. Thisindicates the wild-type alpha-2BAR and thus the polymorphic alpha-2BARis identified.

[0165] To detect the polymorphic site on target nucleic acids encodingthe alpha-2BAR, a primer oligonucleotide complementary to a region ofSEQ ID NO: 2 can be hybridized at the primer's 3′ end to a region up toand including, for example, nucleotide position number 904 of SEQ ID NO:2. If the primer is extended at its 3′ end by a single labeledterminator nucleotide, such as for example the dideoxynucleotide ddCTP,using a polymerase, a detectable signal is produced indicating thecomplementary nucleotide G at nucleotide position 903. This indicatesthe mutant alpha-2BAR shown and thus the polymorphic alpha-2BAR isidentified.

[0166] For example, to detect the polymorphic site on target nucleicacids encoding the alpha-2AAR, a primer oligonucleotide complementary toa region of SEQ ID NO: 24 can be hybridized at the primer's 3′ end to aregion up to and including, for example, nucleotide position number 754of SEQ ID NO: 24. If the primer is extended at its 3′ end by a singlelabeled terminator nucleotide, such as for example the dideoxynucleotideddGTP, using a polymerase, a detectable signal is produced indicatingthe complementary nucleotide C at nucleotide position 753. Thisindicates the wild-type alpha-2AAR shown in FIG. 5A (bolded arrow) andthus the polymorphic alpha-2AAR is identified.

[0167] For example, to detect the polymorphic site on target nucleicacids encoding the alpha-2CAR, a primer oligonucleotide complementary toa segment of SEQ ID NO: 40 can be hybridized at the primer's 3′ end to aregion up to and including, for example, nucleotide position number 972of SEQ ID NO: 40. If the primer is extended at its 3′ end by a singlelabeled terminator nucleotide, such as for example the dideoxynucleotideddGTP, using a polymerase, a detectable signal is produced indicatingthe complementary nucleotide C at nucleotide position 971. Thisindicates the twelve nucleotide deletion shown in FIG. 11B (boldedarrow) and thus the polymorphic alpha-2CAR is identified.

[0168] In some embodiments of the present invention, the oligonucleotideis bound to a solid support prior to the extension reaction. In otherembodiments, the extension reaction is performed in solution and theextended product is subsequently bound to a solid support.

[0169] In an alternate sub-embodiment of GBA, the primer is detectablylabeled and the extended terminator nucleotide is modified so as toenable the extended primer product to be bound to a solid support. Anexample of this would be where the primer is fluorescently labeled andthe terminator nucleotide is a biotin-labeled terminator nucleotide andthe solid support is coated or derivatized with avidin or streptavidin.In such embodiments, an extended primer would thus be enabled to bind toa solid support and non-extended primers would be unable to bind to thesupport, thereby producing a detectable signal dependent upon asuccessful extension reaction.

[0170] Ligase/polymerase mediated genetic bit analysis (U.S. Pat. Nos.5,679,524, and 5,952,174, both herein incorporated by reference) isanother example of a suitable polymerase mediated primer extensionmethod for determining the identity of a nucleotide at a polymorphicsite. Ligase/polymerase GBA utilizes two primers. Generally, one primeris detectably labeled, while the other is designed to be affixed to asolid support. In alternate embodiments of ligase/polymerase GBA,extended nucleotide is detectably labeled. The primers inligase/polymerase GBA are designed to hybridize to each side of apolymorphic site, such that there is a gap comprising the polymorphicsite. Only a successful extension reaction, followed by a successfulligation reaction enables the production of the detectable signal. Themethod offers the advantages of producing a signal with considerablylower background than is possible by methods employing onlyhybridization or primer extension alone.

[0171] The present invention includes an alternate method fordetermining the identity of a nucleotide at a predetermined polymorphicsite in a target polynucleotide. This method is described in Söderlundet al., U.S. Pat. No. 6,013,431, the entire disclosure is hereinincorporated by reference. In this alternate method, the polymorphicsite is interrogated where nucleotide sequence information surrounding apolymorphic site in a target nucleic acid sequence is used to design anoligonucleotide primer that is complementary to a region flanking the 3′or 5′ end of the target polynucleotide, but not including, the variablenucleotide(s) in the polymorphic site of the target polynucleotide. Thetarget polynucleotide is isolated from the biological sample andhybridized to the interrogating primer. In some embodiments of thismethod, following isolation, the target polynucleotide may be amplifiedby any suitable means prior to hybridization to the interrogatingprimer. The primer is extended, using a polymerase, often in thepresence of a mixture of at least one labeled deoxynucleotide and one ormore chain terminating nucleoside triphosphate-precursors (or suitable,analogs). A detectable signal is thereby produced upon incorporation ofthe labeled deoxynucleotide into the primer.

[0172] Cohen, D. et al. (PCT Application W091/02087) describes anotherexample of a suitable method for determining the identity of apolymorphic site, wherein dideoxynucleotides are used to extend a singleprimer by a single nucleotide in order to determine the sequence at adesired locus. Ritterband, M., et al., (PCT Application W095/17676)describes an apparatus for the separation, concentration and detectionof such target molecules in a liquid sample. Cheeseman, P. C. (U.S. Pat.No. 5,302,509) describes a related method of determining the sequence ofa single stranded DNA molecule. The method of Cheeseman employsfluorescently labeled 3′-blocked nucleotide triphosphates with each basehaving a different fluorescent label.

[0173] Wallace et al. (PCT Application W089/10414) describes multiplePCR procedures which can be used to simultaneously amplify multipleregions of a target by using allele specific primers. By using allelespecific primers, amplification can only occur if a particular allele ispresent in a sample.

[0174] Several other suitable primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Bajaj et al. (U.S. Pat. No.5,846,710); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992);Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyrén, P. et al., Anal.Biochem. 208:171-175 (1993)). These methods differ from GBA in that theyall rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidewill result in signals that are proportional to the length of the run(Syvanen, A. -C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)). Such arange of locus-specific signals could be more complex to interpret,especially for heterozygotes, compared to the simple, ternary (2:0, 1:1,or 0:2) class of signals produced by the GBA method.

[0175] Amplification

[0176] In certain embodiments of the present invention, the detection ofpolymorphic sites in a target polynucleotide may be facilitated throughthe use of nucleic acid amplification methods. Such methods may be usedto specifically increase the concentration of the target polynucleotide(i.e., sequences that span the polymorphic site, or include that siteand sequences located either distal or proximal to it). Such amplifiedmolecules can be readily detected by gel electrophoresis, or othermeans.

[0177] The most preferred method of achieving such amplification employsPCR (e.g., Mullis, et al., U.S. Pat. No. 4,965,188), using primer pairsthat are capable of hybridizing to the proximal sequences that define orflank a polymorphic site in its double-stranded form.

[0178] In some embodiments of the present invention, the amplificationmethod is itself a method for determining the identity of a polymorphicsite, as for example, in allele-specific PCR (J. Turki et al., J. Clin.Invest. 95:1635-1641 (1995)). In allele-specific PCR, primer pairs arechosen such that amplification is dependent upon the input templatenucleic acid containing the polymorphism of interest. In suchembodiments, primer pairs are chosen such that at least one primer is anallele-specific oligonucleotide primer. In some sub-embodiments of thepresent invention, allele-specific primers are chosen so thatamplification creates a restriction site, facilitating identification ofa polymorphic site. In other embodiments of the present invention,amplification of the target polynucleotide is by multiplex PCR (Wallaceet al. (PCT Application W089/10414)). Through the use of multiplex PCR,a multiplicity of regions of a target polynucleotide may be amplifiedsimultaneously. This is particularly advantageous in those embodimentswherein greater than a single polymorphism is detected.

[0179] In lieu of PCR, alternative methods, such as the “Ligase ChainReaction” (“LCR”) may be used (Barany, F., Proc. Natl. Acad. Sci.(U.S.A.) 88:189-193 (1991)). LCR uses two pairs of oligonucleotideprobes to exponentially amplify a specific target. The sequences of eachpair of oligonucleotides are selected to permit the pair to hybridize toabutting sequences of the same strand of the target. Such hybridizationforms a substrate for a template-dependent ligase. As with PCR, theresultant product serves as a template in subsequent amplificationcycles, resulting in an exponential amplification of the desiredsequence.

[0180] In accordance with the present invention, LCR can be performedusing oligonucleotides having sequences derived from the same strand,located proximal and distal to the polymorphic site. In one embodiment,either oligonucleotide is designed so as to include the actualpolymorphic site of the polymorphism. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule contains the specificnucleotide in the polymorphic site that is complementary to thepolymorphic site present on the oligonucleotide. In an alternativeembodiment, the oligonucleotides will not include the polymorphic site,such that when they hybridize to the target molecule, a “gap” of atleast one nucleotide is created (see, Segev, D., PCT ApplicationW090/01069 and U.S. Pat. No. 6,004,826). This gap is then “filled” withcomplementary dNTPs (as mediated by DNA polymerase), or by an additionalpair of oligonucleotides. Thus, at the end of each cycle, each singlestrand has a complement capable of serving as a target during the nextcycle and exponential amplification of the desired sequence is obtained.

[0181] The “Oligonucleotide Ligation Assay” (“OLA”) (Landegren, U. etal., Science 241:1077-1080 (1988)) shares certain similarities with LCRand is also a suitable method for analysis of polymorphisms. The OLAprotocol uses two oligonucleotides, which are designed to be capable ofhybridizing to abutting sequences of a single strand of a target. OLA,like LCR, is particularly suited for the detection of point mutations.Unlike LCR, however, OLA results in “linear” rather than exponentialamplification of the target sequence.

[0182] Nickerson, D. A. et al. have described a nucleic acid detectionassay that combines attributes of PCR and OLA (Nickerson, D. A. et al.,Proc. Natl. Acad Sci. (U.S.A.) 87:8923-8927 (1990)). In this method, PCRis used to achieve the exponential amplification of target DNA, which isthen detected using OLA.

[0183] Schemes based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“dioligonucleotide”, thereby amplifying the dioligonucleotide, are known(Wu, D. Y. et al., Genomics 4:560 (1989); Adams, C., W094/03630), andare also suitable methods for the purposes of the present invention.

[0184] One convenient method for identifying genetic polymorphisms iscalled random amplified polymorphic DNA (“RAPD”). It requires no probeDNA and no advance information about the genome of the organism, butuses a set of PCR primers of 8 to 10 bases whose sequence is random. Therandom primers are tried singly or in pairs in PCR reactions, and sincethe primers are short, they often anneal to the target DNA at multiplesites. Some primers anneal in the proper orientation and at a suitabledistance from each other to support amplification of the unknownsequence between them. Among the set of fragments are ones that can beamplified from some genomic DNA samples but not from others, which meansthat the presence or absence of the fragment is polymorphic in thepopulation of organisms. An important feature of RAPDs and otherdetection methods based on PCR amplification is that presence of thefragment is dominant to absence of the fragment. Thus, if one allele (+)supports amplification but the alternative allele (−) does not, then DNAfrom the genotypes +/+ and +/− will support amplification equally well,whereas DNA from the genotype −/− will not support amplification. The+allele is therefore dominant to the −allele in regard to thecorresponding RAPD fragment.

[0185] Other known nucleic acid amplification procedures includetranscription-based amplification systems (Malek, L. T. et al., U.S.Pat. No. 5,130,238; Davey, C. et al., European Patent Application329,822; Schuster et al., U.S. Pat. No. 5,169,766; Miller, H. I. et al.,PCT-Application W089/06700; Kwoh, D. et al., Proc. Natl. Acad Sci.(U.S.A.) 86:1173 Z1989); Gingeras, T. R. et al., PCT ApplicationW088/10315)), or isothermal amplification methods (Walker, G. T. et al.,Proc. Natl. Acad Sci. (U.S.A.) 89:392-396 (1992)) may also be used.

[0186] Gel Migration

[0187] Single strand conformational polymorphism (SSCP; M. Orita et al.,Genomics 5:874-879 (1989);Humphfies et al., In: Molecular Diagnosis ofGenetic Diseases, R. Elles, ed. pp321-340 (1996)) and temperaturegradient gel electrophoresis (TGGE; R. M. Wartell et al., Nucl. AcidsRes. 18:2699-2706 (1990)) are examples of suitable gel nmigration-basedmethods for determining the identity of a polymorphic site. In SSCP, asingle strand of DNA will adopt a conformation that is uniquelydependent of its sequence composition. This conformation is usuallydifferent, if even a single base is changed. Thus, certain embodimentsof the present invention, SSCP may be utilized to identify polymorphicsites, as wherein amplified products (or restriction fragments thereof)of the target polynucleotide are denatured, then run on a non-denaturinggel. Alterations in the mobility of the resultant products are thusindicative of a base change. Suitable controls and knowledge of the“normal” migration patterns of the wild-type alleles may be used toidentify polymorphic variants.

[0188] Temperature gradient gel electrophoresis (TGGE) is a relatedprocedure, except that the nucleic acid sample is run on a denaturinggel. In embodiments of the present invention utilizing TGGE to identifya polymorphic site, the amplified products (typically PCR products) areelectrophoresed over denaturing polyacrylamide gel, wherein thetemperature gradient is optimized for separation of the targetpolynucleotide segments (E. Reihsaus et al., Am. J. Respir. Cell Mol.Biol. 8:334-339 (1993), herein incorporated by reference). This methodis able to detect single base changes in the target polynucleotidesequence.

[0189] Kits of the Present Invention

[0190] The present invention provides diagnostic and therapeutic kitsthat include at least one primer for detecting at least one polymorphismin nucleic acids encoding an alpha-2B, alpha-2A or alpha-2C adrenergicreceptor molecule. Preferably, the kit includes a container having anoligonucleotide comprising a region of SEQ ID NOs: 1 or 2, SEQ ID NOs:24 or 25, or SEQ ID NOs: 40 or 42 or complement thereof for detectingthe polymorphism as described. In one embodiment, the kit includesprimers for amplifying regions of nucleic acids encoding the alpha-2B,alpha-2A or alpha-2C adrenergic receptor molecule where at least one ofthe polymorphisms is found, such as for example SEQ ID NOs: 1 or 2, SEQID NOs: 24 or 25, or SEQ ID NOs: 40 or 42, respectively. In an alternateembodiment, the kit includes allele-specific oligonucleotides, specificfor both mutant and wild-type alleles of at least one polymorphism. Thekit may also contain sources of “control” target polynucleotides, aspositive and negative controls. Such sources may be in the form ofpatient nucleic acid samples, cloned target polynucleotides, plasmids orbacterial strains carrying positive and negative control DNA. Kitsaccording to the invention can include one or more containers, as wellas additional reagent(s) and/or active and/or inert ingredient(s) forperforming any variations on the methods of the invention. Exemplaryreagents include, without limitation, one or more primers, one or moreterminator nucleotides, such as dideoxynucleotides, that are labeledwith a detectable marker. The kits can also include instructions formixing or combining ingredients or use.

[0191] The invention also provides diagnostic and experimental kitswhich include monospecific antibodies that enable the detection,purification and/or separation of alpha-2B, alpha-2A, or alpha-2Cadrenergic receptor molecules or fragments thereof in a specific andreproducible manner. In these kits, the antibodies may be provided withmeans for binding to detectable marker moieties or substrate surfaces.Alternatively, the kits may include the antibodies already bound tomarker moieties or substrates. The kits may further include positiveand/or negative control reagents as well as other reagents for adaptingthe use of the antibodies to particular experimental and/or diagnostictechniques as desired. The kits may be prepared for in vivo or in vitrouse, and may be particularly adapted for performance of any of themethods of the invention, such as ELISA. For example, kits containingantibody bound to multi-well microtiter plates can be manufactured.

[0192] Genotyping and Haplotyping Methods

[0193] The polymorphic sites of the present invention occurring in thepolynucleotide encoding alpha-2B, alpha-2A, or alpha-2C adrenergicreceptor gene can be detected by any of the above methods and used todetermine the genotype. As used herein, the term “genotyping” refers todetermining the presence, absence or identity of a polymorphic site in atarget nucleic acid (identified as SEQ ID NOs: 1 or 2; SEQ ID NOs: 24 or25; or SEQ ID NOs: 40 or 42). Preferably, the genotyping is performed ontwo copies of the alpha-2B, alpha-2A, or alpha-2C adrenergic receptorgene.

[0194] In one embodiment, genotyping involves obtaining a samplecontaining the target nucleic acid, treating the sample to obtain singlestranded nucleic acids, if such nucleic acid is double-stranded, so asto obtain unpaired nucleotide bases spanning the specific position. Ifthe target nucleic acid is single-stranded, this step is not necessary.The sample containing the target nucleic acid is contacted with anoligonucleotide under hybridizing conditions. The oligonucleotide iscapable of hybridizing with a stretch of nucleotide bases present in thetarget nucleic acid, adjacent to the polymorphic site to be identified(e.g., deletion, insertion, mutation, or a single nucleotidepolymorphisms), so as to form a duplex between the oligonucleotide andthe target nucleic acid. When the oligonucleotide is “immediatelyadjacent” the polymorphic site to be identified, the oligonucleotidehybridizes with the target nucleic acid in such a way that either the 3′or 5′ end of the oligonucleotide is complementary to a nucleotide on thetarget nucleic acid that is located immediately 5′ or 3′, respectively,of the polymorphic site to be identified. It is also contemplated hereinthat the oligonucleotide can be a fragment complementary to SEQ ID NO: 1or 2, SEQ ID NO: 24 or 25, or SEQ ID NO: 40 or 42 and not immediatelyadjacent to the polymorphic site to be identified, such that the 3′ endof the oligonucleotide is 1 up to 50, preferably 1 up to 20, nucleotidesupstream or downstream from the polymorphic site to be identified in thetarget nucleic acid.

[0195] As used herein, upstream includes that part of a strand of DNA orRNA molecule that is towards the 5′ end of the polymorphic site or siteof interest. For example, upstream of the polymorphic site of thealpha-2B target polynucleotide (nucleotide positions 901 to 909 of SEQID NO: 1 or 2) includes nucleotide positions 880 to 900. Also, forexample, upstream of the polymorphic site of the alpha-2A targetpolynucleotide (nucleotide position 753 of SEQ ID NO: 24 or 25) includesnucleotide positions 752 to 732. Also, for example, upstream of thepolymorphic site of the alpha-2C target polynucleotide (nucleotidepositions 964 to 975 of SEQ ID NO: 40 or 42) includes nucleotidepositions 951 to 963. Downstream includes that part of a strand of DNAor RNA molecule lying towards the 3′ end of the polymorphic site or siteof interest. For example, downstream of the polymorphic site of thealpha-2B target polynucleotide (nucleotide positions 901 to 909 of SEQID NO: 1 or 2) includes nucleotide positions 910 to 930. Also, forexample, downstream of the polymorphic site of the alpha-2A targetpolynucleotide (nucleotide position 753 of SEQ ID NO: 24 or 25) includesnucleotide positions nucleotide positions 754 to 764. Also, for exampledownstream of the polymorphic site of the alpha-2C target polynucleotide(nucleotide positions 964 to 975 of SEQ ID NO: 40 or 42) includesnucleotide positions 976 to 988.

[0196] In one preferred embodiment of the present invention, to detectthe polymorphic site on target nucleic acids encoding the alpha-2BAR, aprimer oligonucleotide complementary to a region of SEQ ID NO: 1 can behybridized at the primer's 3′ end to a region up to and including, forexample, nucleotide position number 902 of SEQ ID NO: 1. If the primeris extended at its 3′ end by a single labeled terminator nucleotide,such as for example the dideoxynucleotide ddTTP, using a polymerase, adetectable signal is produced indicating the complementary nucleotide Aat nucleotide position 903. This indicates the wild-type alpha-2BAR andthus the polymorphic alpha-2BAR is identified.

[0197] To detect the polymorphic site on target nucleic acids encodingthe alpha-2BAR, a primer oligonucleotide complementary to a region ofSEQ ID NO: 2 can be hybridized at the primer's 3′ end to a region up toand including, for example, nucleotide position number 904 of SEQ ID NO:2. If the primer is extended at its 3′ end by a single labeledterminator nucleotide, such as for example the dideoxynucleotide ddCTP,using a polymerase, a detectable signal is produced indicating thecomplementary nucleotide G at nucleotide position 903. This indicatesthe mutant alpha-2BAR and thus the polymorphic alpha-2BAR is identified.The above described methods are useful in determining the alpha-2BARgenotype or haplotype. In one embodiment, the present invention providesa method of genotyping an alpha-2B adrenergic receptor gene comprising:obtaining a sample having a polynucleotide encoding analpha-2B-adrenergic receptor molecule comprising SEQ ID NOs: 1 or 2 orfragment or complement of the polynucleotide; and detecting in thesample a polymorphic site comprising nucleotide positions 901 to 909 ofSEQ ID Nos: 1 or 2 or fragment or complement thereof.

[0198] In the most preferred embodiment, the present invention includesmethods of genotyping nucleic acids encoding an alpha-2B, alpha-2A, oralpha-2C adrenergic receptor molecule from a sample of an individualwhich includes isolating from the individual, the sample having apolynucleotide encoding the alpha-2B, alpha-2A, or alph-2C adrenergicreceptor molecule identified as SEQ ID NO: 1 or 2, SEQ ID NO: 24 or 25,or SEQ ID NO: 40 or 42, respectively, or fragment or complement thereof;incubating the polynucleotide with at least one oligonucleotide, theoligonucleotide having a nucleotide sequence that is complementary to aregion of the polynucleotide, and which, when hybridized to the regionpermits the identification of the nucleotide present at a polymorphicsite of the polynucleotide, wherein the incubation is under conditionssufficient to allow specific hybridization to occur betweencomplementary nucleic acid molecules; permitting the hybridization tooccur; and identifying the polymorphic site to obtain the genotype ofthe individual. A genotype includes a 5′ to 3′ sequence of nucleotidepair(s) found at one or more polymorphic sites in a locus on a pair ofhomologous chromosomes in an individual.

[0199] In one embodiment of the present invention, to detect thepolymorphic site on target nucleic acids encoding the alpha-2AAR, aprimer oligonucleotide complementary to a region of SEQ ID NO: 24 can behybridized at the primer's 3′ end to a region up to and including, forexample, nucleotide position number 754 of SEQ ID NO: 24. If the primeris extended at its 3′ end by a single labeled terminator nucleotide,such as for example the dideoxynucleotide ddGTP, using a polymerase, adetectable signal is produced indicating the complementary nucleotide Cat nucleotide position 753. This indicates the wild-type alpha-2AARshown in FIG. 5A (bolded arrow) and thus the polymorphic alpha-2AAR isidentified.

[0200] The present invention includes a method for haplotyping analpha-2B adrenergic receptor gene comprising: obtaining a sample havinga polynucleotide encoding an alpha-2B-adrenergic receptor moleculecomprising SEQ ID NOs: 1 or 2 or fragment or complement of thepolynucleotide; detecting in the sample a polymorphic site comprisingnucleotide positions 901 to 909 of SEQ ID NOs: 1 or 2 or fragment orcomplement thereof on one copy of the alpha-2B-adrenergic receptor gene;and determining the identity of an additional polymorphic site on thecopy of the alpha-2B-adrenergic receptor gene.

[0201] As used herein, haplotyping includes determining the identity oftwo or more polymorphic sites in a locus on a single chromosome from asingle individual. Haplotypes include 5′ to 3′ sequence of nucleotidesfound at two or more polymorphic sites in a locus on a single chromosomefrom an individual. The preferred polymorphic sites are discussed above.

[0202] Once the haplotype or genotype is determined in the individual,this information can be compared to any particular alpha-2BAR,alpha-2AAR or alpha-2CAR genotype or haplotype found in a population. Ina preferred embodiment, the alpha-2BAR, alpha-2AAR or alpha-2CARgenotype may also comprise the nucleotide pair(s) detected at one ormore additional alpha-2BAR, alpha-2AAR or alpha-2CAR polymorphic sites.The population may be a reference population, a family population, asame sex population, a population group, a trait population (e.g., agroup of individuals exhibiting a trait of interest such as a medicalcondition or response to a therapeutic treatment i.e. drug). Populationgroups include a group of individuals sharing a common ethno-geographicorigin. Reference populations include a group of subjects or individualswho are predicted to be representative of the genetic variation found inthe general population. Preferably, the reference population representsthe genetic variation in the population at a certainty level of at least85%, preferably at least 90%, more preferably at least 95% and even morepreferably at least 99%.

[0203] Frequency data for such alpha-2BAR, alpha-2AAR or alpha-2CARgenotypes or haplotypes in reference and trait populations are usefulfor identifying an association between a trait and an alpha-2BAR,alpha-2AAR or alpha-2CAR polymorphism, an alpha-2BAR, alpha-2AAR oralpha-2CAR genotype or an alpha-2BAR, alpha-2AAR or alpha-2CARhaplotype. The trait may be any detectable phenotype, including but notlimited to genetic predisposition to a disease or response to atreatment, such as for example, agonist or antagonist. These data can beused to determine a measurable or baseline effect of the agonist orantagonist correlated with the polymorphism, genotype or haplotype.

[0204] In one embodiment, the method of the present invention includesobtaining data on the frequency of the alpha-2BAR, alpha-2AAR, oralpha-2CAR polymorphism, alpha-2BAR, alpha-2AAR or alpha-2CAR genotypeor alpha-2BAR, alpha-2AAR or alpha-2CAR haplotype of interest in areference population as well as in a population exhibiting the trait.Frequency data for one or both of the reference and trait populationsmay be obtained by genotyping or haplotyping each individual in thepopulations using one of the methods described herein. In anotherembodiment, the frequency data for the reference and/or traitpopulations is obtained by accessing previously determined frequencydata, which may be in written or electronic form. For example, thefrequency data may be present in a database that is accessible by acomputer. Once the frequency data is obtained, the frequencies of thealpha-2BAR, alpha-2AAR or alpha-2CAR polymorphism, alpha-2BAR,alpha-2AAR, or alpha-2CAR genotype or alpha-2BAR, alpha-2AAR oralpha-2CAR haplotype of interest are compared in the reference and traitpopulations. If an alpha-2BAR, alpha-2AAR or alpha-2CAR polymorphism,alpha-2BAR, alpha-2AAR, or alpha-2CAR genotype or alpha-2BAR, alpha-2AARor alpha-2CAR haplotype is more frequent in the trait population than inthe reference population to a statistically significant degree, then thetrait is predicted to be associated with that alpha-2BAR, alpha-2AAR oralpha-2CAR polymorphism, alpha-2BAR, alpha-2AAR, or alpha-2CAR genotypeor alpha-2BAR, alpha-2AAR or alpha-2CAR haplotype.

[0205] Transgenic Animals

[0206] The knowledge of the alpha-2A-adrenergic receptor moleculeidentified as amino acids SEQ ID NO: 26 or 27 of the invention, togetherwith the cloning and sequencing of the nucleic acids encoding thealpha-2A-adrenergic receptor molecule identified as SEQ ID NO: 24 or 25,enables other applications of the invention. The knowledge of thealpha-2C-adrenergic receptor molecule identified as amino acids SEQ IDNO: 5 or 7 of the invention, together with the cloning and sequencing ofthe nucleic acids encoding the alpha-2C-adrenergic receptor moleculeidentified as SEQ ID NO: 1 or 3, enables other applications of theinvention. For example, genetically altered animals can be constructedusing techniques, such as transgenesis and gene ablation withsubstitution, to express alpha-2B, alpha-2A or alpha-2C adrenergicreceptor gene products. Various such techniques are known, and certainof these techniques can yield heritability of the transgene. See, e.g.,Pinkert et al. (1995) for an overview of these techniques, and thedocuments cited there for greater detail. Also, the production oftransgenic non-human animals is disclosed U.S. Pat. Nos. 5,175,385,5,175,384, 5,175,838 and 4,736,866 which are incorporated herein byreference.

[0207] Briefly, an animal can be transformed by integration of anexpressible transgene comprising a heterologous alpha-2B, alpha-2A oralpha-2C adrenergic receptor-related nucleic acid sequence into thegenome of the animal. Preferably the transgene is heritable. Such atransgenic animal can then be used as an in vivo model for production ofthe alpha-2b, alpha-2A, or alpha-2C adrenergic receptor molecule in thespecies from which the gene encoding the alpha-2B, alpha-2A, or alpha-2Cadrenergic receptor molecule is derived. Of particular importance, ofcourse, is the development of animal models for human alpha-2B, alpha-2Aor alpha-2C adrenergic receptor activity. Such transgenic animal modelswould express, for example, the mutant alpha-2B, alpha-2A or alpha-2Cadrenergic receptor molecule normally expressed in humans, and would becapable of being used as in vivo pharmacologic models to studypolymorphisms and effects on receptor activity. In gene ablation withsubstitution (also called “hit and run” or “tag and replace”) the murinealpha-2B, alpha-2A or alpha-2C gene is removed and replaced with thehuman wild-type or mutant alpha-2B, alpha-2A or alpha-2C gene.

[0208] Animals suited for transgenic manipulation include domesticatedanimals, simians and humans. Domesticated animals include those of thefollowing species: canine; feline; bovine; equine; porcine; and murine.

[0209] In one exemplary approach a genetically altered test animal isadministered a putative alpha-2B, alpha-2A or alpha-2C adrenergicreceptor agonist or antagonist. Following a time sufficient to produce ameasurable effect in an otherwise untreated animal, binding and activityof the agonist or antagonist is measured by methods known in the art,such as radio-ligand binding assays, adenyly cyclase, MAP kinase orinositol phosphate activity. A lower than normal rate of binding oractivity indicates that the receptor is defective.

[0210] Antibodies

[0211] The present invention provides antibodies raised against thealpha-2A-adrenergic receptor protein identified as SEQ ID NO: 26 or 27or fragment thereof. Such antibodies can bind, preferably specifically,with amino acid position 251 of SEQ ID NO: 26 or 27. These antibodiesform the basis of a diagnostic test or kits. An “antibody” in accordancewith the present specification is defined broadly as a protein thatbinds specifically to an epitope, such as for example the lysine atamino acid position 251 of SEQ ID NO: 27 or fragment thereof. Thepresent invention provides antibodies raised against thealpha-2C-adrenergic receptor protein identified as SEQ ID NO: 28 or 30or fragment thereof. Such antibodies can bind, preferably specifically,with an epitope on SEQ ID NO: 28 or 30 or fragment thereof. Theseantibodies form the basis of a diagnostic test or kits. An “antibody” inaccordance with the present specification is defined broadly as aprotein that binds specifically to an epitope, such as for examplepeptides SEQ ID NO: 29 or 31 or fragment thereof. The antibody may bepolyclonal or monoclonal. Antibodies further include recombinantpolyclonal or monoclonal Fab fragments prepared in accordance with themethod of Huse et al., Science 246, 1275-1281 (1989) and Coligan, J. E.et al. (Eds.) Current Protocols in Immunology, Wiley Intersciences, NewYork, (1999).

[0212] Preparing Antibodies

[0213] Polyclonal antibodies are isolated from mammals that have beeninnoculated with the protein or a functional analog, such as inaccordance with methods known in the art (Coligan, J. E, et al. (Eds.),Current Protocols in imunology,Wiley Intersciences, New York, (1999)).Briefly, polyclonal antibodies may be produced by injecting a hostmammal, such as a rabbit, mouse, rat, or goat, with the protein or afragment thereof capable of producing antibodies that distinguishbetween mutant and wild-type protein. The peptide or peptide fragmentinjected may contain the wild-type sequence or the mutant sequence. Serafrom the mammal are extracted and screened to obtain polyclonalantibodies that are specific to the peptide or peptide fragment.

[0214] The antibodies are preferably monoclonal. Monoclonal antibodiesmay be produced by methods known in the art. These methods include theimmunological method described by Kohler and Milstein in Nature256:495-497 (1975) and by Campbell in “Monoclonal Antibody Technology,The Production and Characterization of Rodent and Human Hybridomas” inBurdon et al. (Eds.), Laboratory Techniques in Biochemistry andMolecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam(1985); and Coligan, J. E, et al. (Eds.), Current Protocols inImmunology,Wiley Intersciences, New York, (1999); as well as therecombinant DNA method described by Huse et al., Science 246:1275-1281(1989).

[0215] In order to produce monoclonal antibodies, a host mammal isinoculated with a peptide or peptide fragment as described above, andthen boosted. Spleens are collected from inoculated mammals a few daysafter the final boost. Cell suspensions from the spleens are fused witha tumor cell in accordance with the general method described by Kohlerand Milstein in Nature 256:495-497 (1975). See also Campbell,“Monoclonal Antibody Technology, The Production and Characterization ofRodent and Human Hybridomas” in Burdon et al. (Eds.), LaboratoryTechniques in Biochemistry and Molecular Biology, Volume 13, ElsevierScience Publishers, Amsterdam (1985) and Coligan, J. E., et al. (Eds.),Current Protocols in Immunology,Wiley Intersciences, New York, (1999)).In order to be useful, a peptide fragment must contain sufficient aminoacid residues to define the epitope of the molecule being detected.

[0216] If the fragment is too short to be immunogenic, it may beconjugated to a carrier molecule. Some suitable carrier moleculesinclude keyhold limpet hemocyanin and bovine serum albumin. Conjugationmay be carried out by methods known in the art (Coligan, J. E. et al.(Eds.) Current Protocols in Immunology, Chapter 9, Wiley Intersciences,New York, (1999)). One such method is to combine a cysteine residue ofthe fragment with a cysteine residue on the carrier molecule.

[0217] Methods for preparing polyclonal and monoclonal antibodies thatexhibit specificity toward single amino acid differences betweenpeptides are described by McCormick et al. in U.S. Pat. No. 4,798,787.These methods are incorporated herein by reference.

[0218] Predicting Individual's Response and Selecting Appropriate Drugs

[0219] In a preferred embodiment of the method of the present invention,the trait of interest is a response exhibited by an individual to sometherapeutic treatment, for example, response to a drug targetingalpha-2BAR, alpha-2AAR or alpha-2CAR or response to a therapeutictreatment for a disease. As used herein the term “response” means any orall of the following: a quantitative measure of the response, noresponse, and adverse response (i.e., side effects).

[0220] In order to deduce a correlation between a response to atreatment and an alpha-2BAR, alpha-2AAR, or alpha-2CAR; alpha-2BAR,alpha-2AAR, or alpha-2CAR genotype or alpha-2BAR, alpha-2AAR, oralpha-2CAR haplotype, the clinician can obtain data on the clinicalresponses exhibited by a population of individuals who received thetreatment, hereinafter the “clinical population”. This clinical data maybe obtained by analyzing the results of a clinical trial that hasalready been run and/or the clinical data may be obtained by designingand carrying out one or more new clinical trials. As used herein, theterm “clinical trial” means any research study designed to collectclinical data on responses to a particular treatment, and includes butis not limited to phase I, phase II and phase III clinical trials.Standard methods are used to define the patient population and to enrollsubjects.

[0221] It is preferred that the individuals included in the clinicalpopulation have been assessed for the clinical characteristics of thedisease of interest. Such clinical characteristics may include symptoms,disease severity, response to therapy and the like. Characterization ofpotential patients could employ a standard physical exam or one or morelab tests.

[0222] The therapeutic treatment (i.e. drug) of interest is administeredto each individual in the trial population and each individual'sresponse to the treatment is measured using one or more predeterminedcriteria. It is contemplated that in many cases, the trial populationwill exhibit a range of responses and that the investigator will choosethe number of responder groups (e.g., none, low, medium, high) made upby the various responses. In addition, the alpha-2BAR, alpha-2AAR, oralpha-2CAR gene for each individual in the trial population is genotypedat least one polymorphic site occurring in the alpha-2BAR, alpha-2AAR,alpha-2CAR, respectively, which may be done before or afteradministering the treatment. As used herein, treatment includes astimulus (i.e., drug) administered internally or externally to anindividual.

[0223] After both the clinical and polymorphism data have been obtained,correlations are created between individual response and the presence ofthe alpha-2BAR, alpha-2AAR or alpha-2CAR polymorphism; alpha-2BAR,alpha-2AAR, or alpha-2CAR genotype; or alpha-2BAR, alpha-2AAR, oralpha-2CAR haplotype. Correlations may be produced in several ways. Inone embodiment, individuals are grouped by their alpha-2BAR, alpha-2AAR,or alpha-2CAR genotype; or alpha-2BAR, alpha-2AAR, or alpha-2CARhaplotype and then the averages and standard deviations of responsesexhibited by the member of each group are calculated. These results arethen analyzed to determine if any observed variation in clinicalresponse between genotype or haplotype groups is statisticallysignificant. Another method involves categorizing the response (e.g.,none, low, medium, high or other such grades) and then assessing whethera particular genotype is more common in one group of responders comparedto another. Statistical analysis methods which may be used are describedin L. D. Fisher and G. vanBelle, “Biostatistics: A Methodology for theHealth Sciences”, Wiley-Interscience (New York) 1993.

[0224] It is also contemplated that the above methods for identifyingassociations between an alpha-2BAR, alpha-2AAR or alpha-2CARpolymorphism; alpha-2BAR, alpha-2AAR, or alpha-2CAR genotype; oralpha-2BAR, alpha-2AAR, or alpha-2CAR haplotype having the alpha-2BAR,alpha-2AAR or alpha-2CAR polymorphism, respectively, may be performedalone, or in combination with genotype(s) and haplotype(s) for one ormore additional genomic regions.

[0225] In the most preferred embodiment, the polymorphisms and moleculesof the present invention can be used to predict an individual'ssensitivity or responsiveness to a pharmaceutical composition or drug,such as for example, an agonists or antagonists. Preferably, theindividual's response to an agonist or antagonist, includes detecting apolymorphic site in the polynucleotide encoding the alpha-2B, thealpha-2A, or alpha-2C adrenergic receptor molecule comprising SEQ IDNOs: 1 or 2, SEQ ID NOs: 24 or 25, or SEQ ID NOs: 40 or 42,respectively, or fragment or complement thereof; and correlating thepolymorphism to a predetermnined response for that particularpolymorphism, haplotype or genotype, thereby predicting the individual'sresponse to the agonist or antagonist. Accordingly, the presentinvention can be employed to guide the clinician in the selection ofappropriate drug(s) or pharmaceutical composition(s). For example,individuals with a polymorphism comprising DEL301-303 of SEQ ID NO: 2 inthe alpha-2BAR molecule are more sensitive to antagonists sinceendogenous agonist activation of the receptor by catecholamines isincreased. Accordingly, with regards to agonists, the response orsensitivity can be predicted to be less for those individuals with theDEL301-303 polymorphism of the alpha-2BAR due to impaired coupling.Using this phenotype, the clinician can administer a higher dose of theagonist to the individual or a different drug altogether. Also, forexample, individuals with a polymorphism comprising lysine at amino acidposition 251 of (SEQ ID NO. 27) in the alpha-2AAR molecule are lesssensitive to antagonists since endogenous agonist activation of thereceptor by catecholamines is increased. Accordingly, with regards toagonists, the response or sensitivity can be predicted to be greater forthose individuals with the polymorphism comprising lysine at amino acidposition 251 of the alpha-2AAR due to impaired coupling. Finally, forexample, individuals with a polymorphism comprising amino acid deletionsGAGP (SEQ ID NO. 45) in the alpha-2CAR molecule are more sensitive toantagonists since agonist activation of the receptor by endogenouscatecholamines is reduced. Accordingly, with regards to agonists, theresponse or sensitivity can be predicted to be less for thoseindividuals with the polymorphism comprising amino acid deletions GAGPof the alpha-2CAR due to impaired coupling.

[0226] Alpha-2B, alpha-2A and alpha-2CAR adrenergic receptor moleculefunction or activity can be measured by methods known in the art. Someexamples of such measurement include radio-ligand binding to thealpha-2B, alpha-2A or alpha-2C adrenergic receptor molecule by anagonist or antagonist, receptor-G protein binding, stimulation orinhibition of adenyly cyclase, MAP kinase, phosphorylation or inositolphosphate (IP3).

[0227] In one embodiment of the present invention, an alpha-2Badrenergic receptor agonist is administered, the agonist activates thealpha-2BAR molecule and G_(i) coupling results in inhibiting adenylylcyclase, and decreased phosphorylation. In this embodiment, the mutantalpha-2BAR shows decreased inhibition of adenylyl cyclase (FIG. 3A-C) ascompared to the wild-type alpha-2BAR with insertion of three glutamicacids (IN301-303) at amino acid position 301 to 303 of SEQ ID NO: 7.Thus, mutant alpha-2BAR has decreased receptor activity or function. ).In another embodiment of the present invention, an alpha-2A adrenergicreceptor agonist is administered, the agonist activates the alpha-2AARmolecule and G_(i) coupling results in inhibiting adenylyl cyclase,stimulation of MAP kinase, or stimulation of IP3. In this embodiment ofthe present invention, the polymorphic or mutant alpha-2AAR showsincreased inhibition of adenylyl cyclase (FIG. 7A-B), increasedstimulation of MAP kinase (FIG. 9B) and increased receptor-G proteinbinding (GTPγ5 binding, FIG. 8) as compared to the wild-type alpha-2AARwith asparagine at amino acid position 251 SEQ ID NO: 26. Thus, thepolymorphic alpha-2AAR has enhanced receptor activity or function. Inanother embodiment of the present invention, the polymorphic alpha-2AARshows increased MAP kinase as compared to the wild-type alpha-2AAR.Thus, mutant or polymorphic alpha-2AAR has enhanced receptor activity orfunction. In another embodiment of the present invention, an alpha-2Cadrenergic receptor agonist is administered, the agonist activates thealpha-2CAR molecule and G_(i) coupling results in inhibiting adenylylcyclase, stimulation of MAP kinase, or stimulation of IP3. In thisembodiment of the present invention, the wild-type alpha-2CAR showsincreased inhibition of adenylyl cyclase stimulation of MAP kinase andstimulation of IP3 as compared to the variant alpha-2CAR with deletionsin amino acids 322-325. Thus, wild-type alpha-2CAR has enhanced receptoractivity or function. In another embodiment of the present invention,the wild-type alpha-2CAR shows increased inositol phosphate accumulationas compared to the variant alpha-2CAR with deletions in amino acids322-325. Thus, wild-type alpha-2CAR has enhanced receptor activity orfunction. Preferably, receptor activity is measured by increased ordecreased adenyly cyclase, MAP kinase, G protein receptor interaction,inositol phosphate and/or phosphorylation. Increased or decreasedadenyly cyclase, MAP kinase, phosphorylation and/or inositol phosphateincludes increases or decreases of preferably from about 10% to about200%, more preferably, from about 20% to about 100%, and mostpreferably, from about 30% to about 60% from normal levels.

[0228] In another embodiment of the present invention, the mutantalpha-2BAR (DEL301-303) showed depressed phosphorylation resulting inloss of short-term agonist-promoted receptor desensitization. Thus, onephenotype of the alpha-2BAR Del301-303 polymorphism is decreasedagonist-promoted phosphorylation that results in a complete loss of theability for the receptor to undergo agonist-promoted desensitization. Asused herein, desensitization includes a decline in response resultingfrom continuous application of agonist or to repeated application ordoses. Clinically, desensitization is exhibited by tachyphylaxis. Asused herein, tachyphylaxis includes rapidly decreasing response to adrug (i.e. agonist or antagonist) or pharmaceutical composition afteradministration of more than one dose.

[0229] For purposes of the present invention, an agonist is any moleculethat activates a receptor. Preferably, the receptor is an alpha-2BAR, analpha-2AAR, or an alpha-2CAR. Preferred agonists include alpha-2B,alpha-2A, or alpha-2C adrenergic receptor agonists, such as for example,epinephrine, norepinephrine, clonidine, oxymetazoline, guanabenz,UK14304, BHT933 and combinations thereof.

[0230] An antagonist is any molecule that blocks a receptor. Preferably,the receptor is an alpha-2BAR, an alpha-2AAR or an alpha-2CAR. Preferredantagonists include alpha-2B, alpha-2A or alpha-2C adrenergic receptorantagonists such as for example, yohimbine, prazosin, ARC 239,rauwolscine, idazoxan, tolazoline, phentolamine and combinationsthereof.

[0231] As used herein a “predetermined response” includes a measurableor baseline effect of the agonist or antagonist correlated with thepolymorphism. For example, individuals with the polymorphism wild-typeinsertion of amino acids 301-303 of the alpha-2B adrenergic receptormolecule display increased alpha agonist promoted coupling to G_(i) andthus increased inhibition of adenylyl cyclase compared to thepolymorphic or mutant alpha-2BAR. Also, the wild-type alpha-2BAR showedincreased phosphorylation resulting in gain of short-termagonist-promoted receptor desensitization when compared to the mutantalpha-2BAR. Also, for example, individuals with the polymorphismwild-type Asn 251 of the alpha-2A adrenergic receptor molecule displaydepressed alpha agonist promoted coupling to G_(i) and thus decreasedinhibition of adenylyl cyclase compared to the polymorphic or mutantalpha-2AAR. Other baseline secondary messenger molecules can be used andcorrelated to the polymorphism, such as MAP kinase and inositolphosphate (See FIGS. 9 and 10). Finally, for example, individuals withthe polymorphism deletions of amino acids 322-325 of the alpha-2Cadrenergic receptor molecule display depressed alpha agonist promotedcoupling to G_(i) and thus decreased inhibition of adenylyl cyclasecompared to the wild-type alpha-2CAR.

[0232] The present invention includes methods for selecting anappropriate drug or pharmaceutical composition to administer to anindividual having a disease associated with alpha-2B, alpha-2C, oralpha-2A adrenergic receptor molecule. The method includes detecting apolymorphic site(s) in the polynucleotide encoding the alpha-2B,alpha-2A, or alpha-2C adrenergic receptor molecule comprising SEQ IDNOs: 1 or 2, SEQ ID NOs: 24 or 25, or SEQ ID NOs: 40 or 42,respectively, or fragment or complement thereof in the individual andselecting the appropriate drug based on the polymorphism or polymorphicsite(s) present. The appropriate drug or pharmaceutical composition canbe determined by those skilled in the art based on the particularpolymorphism identified. For example, individuals with the DEL301-303polymorphism showed depressed phosphorylation resulting in loss ofshort-term agonist-promoted receptor desensitization in the alpha-2BARmolecule. Thus, an agonist or antagonist prone to clinical tachyphylaxiscan be used in patients with the DEL301-303 polymorphism. However, theresponse can be decreased due to the altered coupling of alpha-2BARreceptor such that if this were undesirable, another alpha-2BAR agonistcan be selected, or another drug in a different class, can be employed.Accordingly, with regards to alpha agonists, the response or sensitivitycan be predicted to be less for those individuals with the DEL301-303polymorphism of the alpha-2BAR. This would lead the clinician to“customize” the choice of agonist based on this polymorphism. Theskilled artisan will recognize that individuals with the DEL301-303would be more sensitive to antagonists by virtue of their receptorsbeing partially dysfunctional due to the polymorphism. Thus, theclinician can “customize” the choice of antagonist based on thispolymorphism.

[0233] In another example, individuals with a polymorphism comprisinglysine at amino acid position 251 of (SEQ ID NO. 27) in the alpha-2AARmolecule are less sensitive to antagonists since endogenous agonistactivation of the receptor by endogenous catecholamines is increased.Accordingly, with regards to agonists, the response or sensitivity canbe predicted to be greater for those individuals with the polymorphismcomprising lysine at amino acid position 251 of the alpha-2AAR due toimpaired coupling. This would lead the clinician to select an agonist oralternative drug(s) is indicated.

[0234] In a third example, individuals with a polymorphism comprisingamino acid deletions GAGP (SEQ ID NO. 45) in the alpha-2CAR molecule aremore sensitive to antagonists since agonist-receptor binding is reduced.Therefore, for example, identification of the polymorphism deletions ofamino acids 322-325 of the alpha-2C adrenergic receptor molecule in theindividual would lead the clinician to select alternative drug(s) orincrease the dose of the agonist.

[0235] The present invention also contemplates adjusting or changing thedosing regimen of the drug or pharmaceutical composition based on theinsertion or deletion present at amino acid positions 301 to 303. Forexample, individuals with DEL301-303 genotype do not undergodesensitization or tachylphylaxis with the administration of repeateddoses over time. Thus, the clinician would not expect the pharmacologiceffect of the drug to wane over time. An accelerated dosing regimencould be used in the individual with the DEL301-303 polymorphism withoutthe need for slowly increasing the dose, as would be required in thosewith the wild-type receptor (IN301-303). Accordingly, individuals withthe wild-type genotype (IN301-303) undergo desensitization ortachylphylaxis with the administration of repeated doses over time.Thus, the clinician would expect the pharmacologic effect of the drug towane over time and the clinician would need to slowly increase the doseover time.

[0236] As used herein, “appropriate pharmaceutical composition” includesat least one drug that increases therapeutic efficacy of the drug basedon a patient population with a particular disease. Each population willtypically have a unique characteristic response to the drug. Knowledgeof the efficacies of two or more drugs in treating individuals withdifferent genetic variations provides the opportunity to select the drugeffective in treating a large percentage of the total population ofindividuals while maintaining little or no toxicity.

[0237] For the purposes of the present invention, “correlating thepolymorphism with a predetermine response” includes associating thepredetermined response with the polymorphism that occurs at a higherallelic frequency or rate in individuals with the polymorphism thanwithout. Correlation of the polymorphism with the response can beaccomplished by bio-statistical methods known in the art, such as forexample, Chi-squared tests or other methods described in L. D. Fisherand G. vanBelle, Biostatistics: A Methodology for the Health Sciences,Wiley-Interscience (New York) 1993.

[0238] For example, DEL301-303 polymorphism is more common in Caucasiansthan African-Americans, with allele frequencies of 0.31 and 0.12,respectively. The polymorphism results in depressed phosphorylationcausing a loss of short-term agonist-promoted receptor desensitizationin the alpha-2BAR molecule.

[0239] In another example, the lysine 251 polymorphism which results ina gain in alpha-2AAR function occur at a lower rate in Caucasians. Incontrast, the allelic frequency, is ˜10-fold higher inAfrican-Americans. In a third example, polymorphisms resulting in adefective alpha-2CAR molecule occur at a lower rate in Caucasians withan allele frequency of 0.040. In contrast, the frequency is ˜10-foldhigher (0.381) in African-Americans (Table 5). Therefore, an appropriatedrug can be selected based upon the individual's pharmaco-ethnogenetics.

[0240] For the purposes of the present specification, drugs andpharmaceutical compositions are used interchangeably. Drugs orpharmaceutical compositions contemplated by the present inventioninclude therapeutic compounds such as an analgesic drug, an anestheticagent, an anorectic agent, an anti-anemia agent, an anti-asthma agent,an anti-diabetic agent, an antihistamine, an anti-inflammatory drug, anantibiotic drug, an antimuscarinic drug, an anti-neoplastic drug, anantiviral drug, a cardiovascular drug, a central nervous systemstimulant, a central nervous system depressant, an anti-depressant, ananti-epileptic, an anxyolitic agent, a hypnotic agent, a sedative, ananti-psychotic drug, a beta blocker, a hemostatic agent, a hormone, avasodilator and a vasoconstrictor.

[0241] Preferred drugs include the alpha agonists and alpha antagonists.Most preferred drugs include the alpha-2B, alpha-2A, and alpha-2Cagonists and alpha-2B, alpha-2A, alpha-2C antagonists. According to theinvention, pharmaceutical compositions or drugs comprising one or moreof the therapeutic compounds described above, and a pharmaceuticallyacceptable carrier or excipient, may be administered to an individualpredisposed or having the disease as described, orally, rectally,parenterally, intrasystemically, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),bucally, or as an oral or nasal spray. By “pharmaceutically acceptablecarrier” is meant a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

[0242] Pharmaceutical compositions used in the methods of the presentinvention for parenteral injection can comprise pharmaceuticallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), carboxymethylcellulose andsuitable mixtures thereof, vegetable oils (such as olive oil), andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

[0243] The compositions used in the present methods may also containadjuvants such as preservatives, wetting agents, emulsifying agents, anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents such as sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

[0244] In some cases, in order to prolong the effect of the drugs, it isdesirable to slow the absorption from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

[0245] Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

[0246] The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

[0247] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compounds are mixed with at least one item pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agents.

[0248] Solid compositions of a similar type may also be employed asfillers in soft and hardfilled gelatin capsules using such excipients aslactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0249] The solid dosage forms of tablets, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

[0250] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

[0251] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0252] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

[0253] Topical administration includes administration to the skin ormucosa, including surfaces of the lung and eye. Compositions for topicaladministration, including those for inhalation, may be prepared as a drypowder which may be pressurized or non-pressurized. In nonpressurizedpowder compositions, the active ingredients in finely divided form maybe used in admixture with a larger-sized pharmaceutically acceptableinert carrier comprising particles having a size, for example, of up to100 μm in diameter. Suitable inert carriers include sugars such aslactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 μm.

[0254] Alternatively, the composition or drugs may be pressurized andcontain a compressed gas, such as nitrogen or a liquefied gaspropellant. The liquefied propellant medium and indeed the totalcomposition is preferably such that the active ingredients do notdissolve therein to any substantial extent. The pressurized compositionmay also contain a surface active agent. The surface active agent may bea liquid or solid non-ionic surface active agent or may be a solidanionic surface active agent. It is preferred to use the solid anionicsurface active agent in the form of a sodium salt.

[0255] A further form of topical administration is to the eye. Theactive compounds are delivered in a pharmaceutically acceptableophthalmic vehicle, such that the compounds are maintained in contactwith the ocular surface for a sufficient time period to allow thecompounds to penetrate the corneal and internal regions of the eye, asfor example the anterior chamber, posterior chamber, vitreous body,aqueous humor, vitreous humor, cornea, iris/cilary, lens, choroid/retinaand sclera. The pharmaceutically acceptable ophthalmic vehicle may, forexample, be an ointment, vegetable oil or an encapsulating material.

[0256] Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the active compounds withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax which are solid at roomtemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the drugs.

[0257] The compositions used in the methods of the present invention canalso be administered in the form of liposomes. As is known in the art,liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolizable lipid capable of formingliposomes can be used. The present compositions in liposome form, inaddition to one or more of the active compounds described above, cancontain stabilizers, preservatives, excipients and the like. Thepreferred lipids are the phospholipids and the phosphatidyl cholines(lecithins), both natural and synthetic.

[0258] Typical dosages and durations of treatment are as described inclinician's textbooks such as Physician's Desk Reference 2000,incorporated herein by reference, and will be familiar to physicians andother practitioners in the art.

[0259] The above methods of the present invention can be used in vivo,in vitro, and ex vivo, for example, in living mammals as well as incultured tissue, organ or cellular systems. Mammals include, forexample, humans, as well as pet animals such as dogs and cats,laboratory animals, such as rats and mice, hamsters and farm animals,such as horses and cows. Tissues, as used herein, are an aggregation ofsimilarly specialized cells which together perform certain specialfunctions. Cultured cellular systems include any cells that express thealpha-2BAR, alpha-2AAR or alpha-2CAR molecule, such as pre and postsynaptic neurons in the brain or any cell transfected with the alpha-2B,alpha-2A, or alpha-2C gene.

[0260] Having now generally described the invention, the same may bemore readily understood through the following reference to the followingexamples, which are provided by way of illustration and are not intendedto limit the present invention unless specified.

EXAMPLES

[0261] Examples 1-6 relate to the alpha-2BAR. Examples 7-14 relate tothe alpha-2AAR. Examples 15-21 relate to the alpha-2CAR.

[0262] Examples 1-6 below describe a polymorphic variant of the humanalpha-2BAR which includes a deletion of three glutamic acids (residues301-303) in the third intracellular loop. This polymorphism was found tobe common in Caucasians (31%) and to a lesser extent inAfrican-Americans (12%). The consequences of this deletion were assessedby expressing wild-type and the Del301-303 receptors in CHO and COScells. Ligand binding was not affected while a small decrease incoupling to the inhibition of adenylyl cyclase was observed with themutant. The deletion occurs within a stretch of residues which isthought to establish the milieu for agonist-promoted phosphorylation anddesensitization of the receptor by G-protein coupled receptor kinases(GRKs). Agonist-promoted phosphorylation studies carried out in cellsco-expressing the alpha-2BARs and GRK2 revealed that the Del301-303receptor displayed -56% of wild-type phosphorylation. Furthermore, thedepressed phosphorylation imposed by the deletion was found to result ina complete loss of short-term agonist-promoted receptor desensitization.Thus the major phenotype of the Del301-303 alpha-2BAR is one of impairedphosphorylation and desensitization by GRKs, and thus the polymorphismsrenders the receptor incapable of modulation by a key mechanism ofdynamic regulation.

Example 1

[0263] Polymorphism Detection

[0264] The nucleic acid sequence encoding the third intracellular loopof the human alpha-2BAR (GenBank assession #AF009500, SEQ ID NO: 1) wasexamined for polymorphic variation by performing polymerase chainreactions (PCR) to amplify this portion of the cDNA from genomic DNAderived from blood samples. In this application the adenine of theinitiator ATG codon of the open reading frame of the receptor isdesignated as nucleotide 1 and amino acid 1 is the encoded methionine.The human receptor consists of 450 amino acids . For initialexamination, DNA from 39 normal individuals was utilized. Twooverlapping fragments encompassing the third intracellular loop regionwere generated using the following primers pairs: fragment 1 (534 bp),5′-GCTCATCATCCCTTTCTCGCT-3′ (sense) SEQ ID NO: 13 and5′-AAAGCCCCACCATGGTCGGGT-3′ (antisense) SEQ ID NO: 14 and fragment 2(588 bp), 5′-CTGATCGCCAAACGAGCAAC-3′ (sense) SEQ ID NO: 15 and5′-AAAAACGCCAATGACCACAG-3′ SEQ ID NO: 16 (antisense). The 5′ end of eachsense and antisense primer also contained sequences corresponding to theM13 forward (5′-TGTAAAACGACGGCCAGT-3′) SEQ ID NO: 17 and M13 reverse(5′-CAGGAAACAGCTATGACC-3′) SEQ ID NO: 18 universal sequencing primers,respectively. The PCR reactions consisted of ˜100 ng genomic DNA, 5 pmolof each primer, 0.8 mM dNTPs, 10% DMSO, 2.5 units Platinum taq™ DNApolymerase (Gibco/BRL), 20 uL 5×buffer J (Invitrogen) in a 100 μlreaction volume. Reactions were started by an initial incubation at 94°C. for four minutes, followed by 35 cycles of 94° C. for 30 seconds, 58°C. (fragment 1) or 60° C. (fragment 2) for 30 seconds, and 72° C. forone minute, followed by a final extension at 72° C. for seven minutes.PCR reactions were purified using the QIAquick™ PCR purification system(Qiagen), and automated sequencing of both strands of each PCR productwas performed using Applied Biosystems 370 sequencer using dye primermethods. As discussed, a 9 bp in frame deletion at nucleotide positions901to 909 occuring in SEQ ID NO: 2 was detected which resulted in a lossof three glutamic acid residues at amino acid positions 301-303. Thus,this polymorphism was denoted Del301-303. Of note, previous reports haveidentified this polymorphism (21, 22). Heinonen et al. refer to thepolymorphism as Del 297-299 or DEL 298-300 (which may be a numberingerror) while Baldwin et al. refer to it as a 9-base in-frame deletioncorresponding loss of 3 glutamic acid residues. No other nonsynonymousor synonymous polymorphisms were identified. PCR amplification of 209and 200 bp fragments encompassing this polymorphic region allowedscreening of additional DNA samples whose genotypes were distinguishedby size when run on 4% Nuseive agarose gels. PCR conditions were thesame as described above except that buffer F was used with the followingprimers: 5′-AGAAGGAGGGTGTTTGTGGGG-3′ (sense) SEQ ID NO: 19 and5′-ACCTATAGCACCCACGCCCCT-3′ (antisense) SEQ ID NO: 20.

Example 2

[0265] Constructs and Cell Transfection

[0266] To create the polymorphic alpha-2BAR construct, a 1585 bp PCRproduct encompassing the alpha-2BAR gene was amplified from a homozygousdeletion individual using the following primers:5′-GGCCGACGCTCTTGTCTAGCC-3′ (SEQ ID NO: 21) and5′-CAAGGGGTTCCTAAGATGAG-3′ (SEQ ID NO: 22). This fragment was digestedand subcloned into the Xcm I and BamH I sites of the wild-typealpha-2BAR sequence in the expression vector pBC12BI (17). The integrityof the construct was verified by sequencing. Chinese hamster ovary cells(CHO-K1) were stably transfected by a calcium phosphate precipitationtechnique as previously described using 30 μg of each receptor constructand 0.5 μg of pSV₂neo to provide for G418 resistance (23). Selection ofpositive clones was carried out in 1.0 mg/ml G418 and expression of thealpha-2BAR from individual clonal lines was determined by radioligandbinding as described below. Several clonal lines with matched expressionlevels between 500-1000 fmol/mg were utilized as indicated. Cells weregrown in monolayers in Ham's F-12 medium supplemented with 10% fetalcalf serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 80 μg/mlG418 (to maintain selection pressure) at 37° C. in a 5% CO₂ atmosphere.For phosphorylation experiments, receptors were epitope tagged with theinfluenza hemagglutinin nonopeptide YPYDVPDYA (SEQ ID NO: 23) at theamino terminus. This was accomplished by constructing vectors using theabove constructs with insertions of in-frame sequence encoding thepeptide using PCRs essentially as previously described (24). Taggedreceptors were expressed at ˜15 pmol/mg, along with GRK2 (βARK1), inCOS-7 cells using a DEAE Dextran technique as described (16).

Example 3

[0267] Adenylyl Cyclase Activities

[0268] Alpha-2BAR inhibition of adenylyl cyclase was determined inmembrane preparations from CHO cells stably expressing the two receptorsusing methods similar to those previously described (25). Briefly, cellmembranes (˜20 μg) were incubated with 27 μM phosphoenolpyruvate, 0.6 μMGTP, 0.1 mM cAMP, 0.12 mM ATP, 50 μg/ml myokinase, 0.05 mM ascorbic acidand 2 μCi of [α-32P]ATP in a buffer containing 40 mM HEPES, pH 7.4, 1.6mM MgCl2 and 0.8 mM EDTA for 30 minutes at 37° C. Activities weremeasured in the presence of water (basal), 5 μM forskolin, and 5 μMforskolin with the indicated concentrations of agonists. Reactions wereterminated by the addition of a stop solution containing excess ATP andcAMP and ˜100,000 dpm of [3H]cAMP. Labeled cAMP was isolated by gravitychromatography over alumina columns with [3H]cAMP used to quantitatecolumn recovery. Results are expressed as percent inhibition offorskolin stimulated activity. For desensitization experiments, cellswere pretreated for 30 min at 37° with media alone or with mediacontaining 10 μM norepinephrine, placed on ice, and washed five timeswith cold PBS prior to membrane preparation. Desensitization ofalpha-2BAR is manifested by a shift to the right in the dose-responsecurve for the inhibition of adenylyl cyclase (i.e., increase in EC50)without a significant change in the maximal response (17-19). Toquantitate the magnitude of this desensitization, the inhibitoryresponse under control conditions at a submaximal concentration ofagonist (the EC50) in the assay was determined from the curve andcompared to the response to this same concentration from membranesderived from cells exposed to norepinephrine. This method has beenpreviously validated (26) in several G protein coupled receptor systems.

Example 4

[0269] Radioligand Binding

[0270] Expression of mutant and wild-type alpha-2BAR was determinedusing saturation binding assays as described (25, 27) with [³H]yohimbineor [¹²⁵I]aminoclonidine with 10 μM phentolamine or 10 μM yohimbine,respectively, used to define nonspecific binding. For competitionstudies, membranes were incubated in 50 mM Tris-HCL, pH 7.4, 10 mMMgSO₄, 0.5 mM EDTA with 2.0 nM [³H]yohimbine and 16 concentrations ofthe indicated competitor in the absence or presence of guaninenucleotide for 30 minutes at 25° C. Reactions for the above radioligandbinding studies were terminated by dilution with 4 volumes of ice cold10 mM Tris-HCL, pH 7.4 buffer and vacuum filtration over Whatmann GF/Cglass fiber filters.

Example 5

[0271] Intact Cell Receptor Phosphorylation

[0272] Transiently transfected COS-7 cells expressing equivalent levelsof each receptor were grown to confluence and incubated with [³²P]orthophosphate (˜4 mCi/100 mm plate) for 2 h at 37° C. in 5% CO₂atmosphere. Cells were then incubated in the presence or absence of 100μM norepinephrine for 15 min, washed 5 times with ice-cold PBS,solubilized in 1 ml of a buffer containing 1% Triton-X 100, 0.05% SDS, 1mM EDTA, and 1 mM EGTA in PBS, by rotation in a microcentrifuge tube for2 h at 4° C. This and all subsequent steps included the proteaseinhibitors benzamidine (10 μg/ml), soybean trypsin inhibitor (10 μg/ml),aprotinin (10 mg/ml), and leupeptin (5 μg/ml) and the phosphataseinhibitors sodium fluoride (10 mM) and sodium pyrophosphate (10 mM). (Aseparate flask was scraped in 5 mM Tris/2 mM EDTA and membranes preparedfor radioligand binding as described above.) Unsolubilized material wasremoved by centrifugation at 40000 g at 4° C. for 10 min. The HA epitopetagged alpha-2BARs were immunoprecipitated using an anti-HA highaffinity monoclonal antibody (Roche) as previously described (24).Briefly, solubilized material was preincubated for 2 h at 4° C. withprotein G Sepharose beads to remove nonspecific binding. The supernatantwas then incubated with protein G Sepharose beads and a 1:200 dilutionof antibody for 18 h at 4° C. Following immunoprecipitation, the beadswere washed 3 times by centrifugation and resuspension, and thenincubated at 37° C. for 1 hour in SDS sample buffer. Proteins in thesupernatant were then fractionated on a 10% SDS-polyacrylamide gel withequal amounts of receptor (based on radioligand binding) loaded in eachlane. Signals were visualized and quantitated using a Molecular DynamicsPhosphorImager with ImageQuant™ Software.

Example 6

[0273] Protein Determination, Adenylyl Cyclase and Radioligand BindingAssay and Genotype

[0274] Protein determinations were by the copper bicinchoninic acidmethod (28). Data from adenylyl cyclase and radioligand binding assayswere analyzed by iterative least-square techniques using Prizm™ software(GraphPad, San Diego, Calif.). Agreement between genotypes observed andthose predicted by the Hardy-Weinberg equilibrium was assessed by aChi-squared test with one degree of freedom. Genotype comparisons wereby Fisher's exact test. Comparisons of results from biochemical studieswere by t-tests and significance was considered when p<0.05. Data areprovided as means±standard errors.

Results and Discussion of Examples 1-6

[0275] Sequence analysis of the third intracellular loop of thealpha-2BAR gene from 78 chromosomes revealed a single sequence variant.This consisted of an in-frame 9 bp deletion (GAAGAGGAG, SEQ ID NO: 3)beginning at nucleotide 901 of SEQ ID NO: 1 (FIG. 1a) that results inloss of three glutamic acid residues at amino acid positions 301-303 (SEQ ID NO: 11) of the third intracellular loop of the receptor (FIG. 2).Using the rapid detection method (FIG. 1b), allele frequencies weredetermined in a larger population of apparently normal Caucasians andAfrican-Americans. The frequencies of the wild-type and the Del301-303(mutant) polymorphic alpha-2BAR are shown in Table 1.

[0276] The deletion polymorphism is more common in Caucasians thanAfrican-Americans, with allele frequencies of 0.31 and 0.12,respectively. The distribution of homozygous and heterozygous alleles ineither population was not different than that predicted from theHardy-Weinberg equilibrium (p>0.8).

[0277] The consequences of this polymorphism on ligand binding andreceptor function were evaluated by stably expressing the humanwild-type alpha-2BAR and the Del301-303 receptor in CHO cells (shown inTable 8)

[0278] Saturation radioligand binding studies revealed a small butstatistically significant lower affinity for the alpha-2BAR antagonist[³H]yohimbine for Del301-303 compared to the wild-type receptor(K_(d)=5.1±0.2 vs 3.8±0.3 nM, respectively, n=5, p<0.05). Agonist(epinephrine) competition binding experiments carried out in thepresence of GppNHp revealed a small increase in the K_(i) for thepolymorphic receptor (285±8.7 vs 376±66 nM, n=5, p<0.05). In similarstudies carried out in the absence of guanine nucleotide, two-site fitswere obtained for both receptors with no differences in the K_(L) or thepercentage of receptors in the high affinity state (%R_(H), Table 8).However, a trend towards an increased K_(H) was observed with theDel301-303 mutant. These results prompted additional studies with thepartial agonist radioligand [¹²⁵I]-aminoclonidine. Saturation bindingstudies (in the absence of GppNHp) with concentrations of the ligandfrom 0.2-4 nM revealed a single site with a K_(d)˜1 nM as reported byothers (27). Comparison of the wild-type alpha-2BAR and the Del301-303receptor revealed essentially identical K_(d)s for [¹²⁵I]-aminoclonidine(1.33±0.12 vs 1.22±0.07 nM, respectively). Taken together, the datasuggest that there is little, if any, effect of the deletion in thethird intracellular loop on the conformation of the ligand bindingpocket within the transmembrane spanning domains.

[0279] To address the functional consequences of the mutation, studiesexamining agonist-promoted inhibition of forskolin stimulated adenylylcyclase activities were carried out in lines expressing the wild-typealpha-2BAR and the Del301-303 receptor at densities of 626±54 and 520±82fmol/mg (n=7, p>0.05). The results of these studies are shown in Table9.

[0280] As can be seen, the Del301-303 receptor displayed less inhibitionof adenylyl cyclase (23.4±2.2%) compared to wild-type alph-2BAR(28.5±1.6%, p<0.05). Furthermore, the polymorphic receptor had a greaterEC₅₀ (19.6±5.5 vs 7.9±2.1 nM, p<0.01). Thus, the loss of the threeglutamic acids in the third intracellular loop, which is known tocontain regions important for G-protein coupling, results in a modestdecrease in agonist-mediated receptor function.

[0281] The deletion polymorphism occurs in a highly acidic stretch ofamino acids (EDEAEEEEEEEEEEEE, SEQ ID NO: 9) within the thirdintracellular loop of alpha-2BAR (FIG. 2). The structural importance ofthis region has been previously assessed and shown to be critical forshort-term agonist-promoted receptor phosphorylation leading todesensitization (18). These data and reports by others (29) suggest thatthis acidic environment is necessary for receptor phosphorylation byGRKs. Therefore, to investigate the consequences of this deletionpolymorphism on receptor desensitization, agonist-promoted inhibition ofadenylyl cyclase activity was determined in membranes from CHO cellsexpressing the wild-type and Del301-303 receptor after pretreatment withnorepinephrine. In these experiments, cells were incubated with mediaalone or media containing agonist (10 μM norepinephrine) for 30 min andextensively washed, membranes prepared, and agonist-mediated inhibitionof forskolin stimulated adenylyl cyclase activity was determined. Asdescribed previously (17) and shown in FIG. 3 and Table 9,desensitization of wild-type alpha-2BAR expressed in CHO cells ismanifested by an increase in the EC₅₀ for agonist-mediated inhibition ofadenylyl cyclase. Analysis of composite curves derived from fourindependent experiments shows an increase from 7.4 μM to 29.4 μM for thewild-type alpha-2BAR. In contrast, there was no change in the EC₅₀ forthe deletion receptor following agonist pretreatment (29.5 μM versus31.2 μM). Desensitization was quantitated by examining adenylyl cyclaseactivities at a submaximal concentration of agonist (the EC₅₀ for thecontrol condition). At this concentration, wild-type alpha-2BARinhibited adenylyl cyclase activity by 16.5±3.9%; with agonistpreexposure, inhibition at this same concentration of agonist was7.6±2.3%, (n=4, p<0.05, FIG. 3c), amounting to ˜54% desensitization ofreceptor function. Submaximal inhibition of adenylyl cyclase for theDel301-303 receptor, however, was not different between control andagonist-treated cells (17.1±3.0% vs 15.9±1.7%, n=4, p=ns). In anothertwo cell lines with matched expression of ˜600 fmol/mg, the samedesensitization phenotypes for wild-type and the Del301-303 polymorphicreceptor were observed (data not shown).

[0282] We next performed whole cell phosphorylation studies of thewild-type and polymorphic alpha-2BAR under the same conditions as thoseused for desensitization. We hypothesized that agonist-promotedphosphorylation would be decreased in the polymorphic receptor. However,given that this receptor displays rightward-shifted dose response curvesfor inhibition of adenylyl cyclase at baseline, we also considered thepossibility that the receptor is significantly phosphorylated in thebasal state. Studies were carried out in cells co-transfected with thereceptor and GRK2 (βARK1), a strategy that we have previously shown tobe useful in identifying receptor-GRK interactions (30). The results ofa representative study are shown in FIG. 4a and mean results from fourexperiments in FIG. 4b. The wild-type alpha-2BAR underwent a 5.84±0.49fold increase in phosphorylation with agonist exposure. In contrast,while the Del301-303 receptor displayed some degree of agonist-promotedphosphorylation, the extent was clearly less (3.28±0.24 fold, p<0.05compared to wild-type). Basal phosphorylation was equivalent between thetwo receptors.

[0283] It is interesting to note that this partial loss ofphosphorylation results in a receptor that fails to undergo any degreeof functional desensitization. While it might seem reasonable to assumethat such phosphorylation would be associated with some degree ofdesensitization, several previous studies with the α_(2A)- andalpha-2BAR subtypes indicate that full (i.e., wild-type) phosphorylationis necessary for the desensitization process (16, 18, 24). For theα_(2A)AR, we have shown that four serines in the third intracellularloop are phosphorylated after agonist exposure (16). Removal of serinesby alanine substitution mutagenesis results in a proportional decreasein phosphorylation. Such partial phosphorylation (compared towild-type), however, was found to be insufficient to cause anydetectable desensitization. In a previous study of the alpha-2BAR, wedeleted the entire aforementioned acidic region (18). Agonist-promotedphosphorylation was reduced by ˜50% in this mutant, and desensitizationwas ablated. These results are entirely consistent with the currentwork, where a restricted substitution resulted in a decrease inphosphorylation and a complete loss of desensitization. Finally, we havealso recently shown that a chimeric alpha-2A/alpha-2CAR which undergoesagonist-promoted phosphorylation, fails to exhibit desensitization (24).Taken together with our current work, these results indicate that theconformation of the third loop evoked by GRK mediated phosphorylationwhich provides for the binding of arrestins (which is the ultimate stepthat imparts uncoupling) is highly specific. Thus a precisephosphorylation-dependent conformation is apparently required forarrestin binding to alpha-2AR and subsequent functional desensitization.Perturbations of the milieu can thus have significant functionalconsequences, as occurs with the Del301-303 polymorphic alpha-2BAR.

[0284] Thus the major signaling phenotypes of the alpha-2BAR Del301-303polymorphism is one of decreased agonist-promoted phosphorylation whichresults in a complete loss of the ability for the receptor to undergoagonist-promoted desensitization and a decrease in receptor coupling.The potential physiologic consequences of the polymorphism could berelated to either or both of the above phenotypes. A receptor that failsto undergo desensitization would be manifested as static signalingdespite continued activation of the receptor by endogenous or exogenousagonist. Such a lack of regulation by agonist may perturb the dynamicrelationship between incoming signals and receptor responsiveness thatmaintains homeostasis under normal or pathologic conditions. Recently,Gavras and colleagues (14) have shown that alpha-2B−/+ mice fail todisplay a hypertensive response to salt loading after subtotalnephrectomy. Thus a polymorphic alpha-2BAR that fails to desensitize(i.e., does not display regulatable function) may predispose tosalt-sensitive hypertension. Regarding the therapeutic response toalpha-2AR agonists, the phenotype of the Del301-303 receptor indicatesthat individuals with this polymorphism would display littletachyphylaxis to continued administration of agonists. In addition, theinitial response to agonist would also be reduced based on the somewhatdepressed coupling of the Del301-303 receptor.

[0285] Until recently, it has been difficult to differentiate alpha-2BARfunction from the other two subtypes in physiologic studies. With thedevelopment of knock-out mice lacking each α₂AR subtype (5, 6, 13, 31),certain functions can now be definitively attributed to specificsubtypes. Characterization of the alpha-2BAR knock-out mouse hasindicated that the alpha-2BAR subtype is expressed on vascular smoothmuscle and is responsible for the hypertensive response to α₂AR agonists(13). This indicates that vascular alpha-2BAR contribute to overallvascular tone and thus participate in systemic blood pressureregulation. This role may be more important, though, during adaptiveconditions, such as salt loading, since resting blood pressure is normalin the heterozygous alpha-2B −/+ mice (14). Whether the alpha-2B −/−mice have altered resting blood pressures has not been studied in detaildue to high perinatal lethality of the homozygous knockout (13).However, neither the region of chromosome 2 near the alpha-2BAR codingsequence, nor the deletion polymorphism, have been linked or associatedwith hypertension (21, 22, 32). No studies, though, have assessedwhether the polymorphism is associated with salt-sensitive hypertensionor other phenotypes, or the response to alpha-2BAR agonist.

[0286] In summary, we have delineated the signalling phenotype of apolymorphism of the alpha-2BAR that results in a deletion of threeglutamic acids in the third intracellular loop of the receptor. Thepolymorphism is prevalent in the human population, with a frequency thatis −2 fold greater in Caucasians as compared to African-Americans. Thepolymorphic receptor displays wild-type agonist binding affinity but asmall decrease in function in the resting state. The major phenotype,though, is a significant decrease in agonist-promoted phosphorylation byGRKs, which results in a receptor that fails to display agonist-promoteddesensitization. To our knowledge this is the first polymorphism of anyG-protein coupled receptor to affect GRK-mediated phosphorylation.

[0287] Examples 7-14 below describe a single nucleotide polymorphismoccuring in nucleic acids encoding the alpha-2AAR molecule. Thispolymorphism results in an Asn to Lys substitution at amino acid 251 ofthe third intracellular loop of the alpha-2AAR molecule. The frequencyof Lys251 was 10-fold greater in African-Americans compared toCaucasians, but was not associated with essential hypertension. Todetermine the consequences of this substitution, wild-type and Lys251receptors were expressed in CHO and COS-7 cells. Expression, ligandbinding, and basal receptor function were unaffected by thesubstitution. However, agonist-promoted [³⁵S]GTPγS binding was ˜40%greater with the Lys251 receptor. In studies of agonist-promotedfunctional coupling to G_(i), the polymorphic receptor displayedenhanced inhibition of adenylyl cyclase (60±4.4 vs 46±4.1% inhibition)and markedly enhanced stimulation of MAP kinase (57±9 vs 15±2 foldincrease over basal) compared to wild-type alpha-2AAR. This enhancedagonist function was observed with catecholamines, azepines andimadazolines. In contrast, agonist stimulation of phospholipase C wasnot different between the two receptors. Unlike previously describedvariants of G protein coupled receptors where the minor species causeseither a loss of function or increased non-agonist function, Lys251alpha-2AAR can represent another class of polymorphism whose phenotypeis a gain of agonist-promoted function.

Example 7

[0288] Polymorphism Detection

[0289] The intronless wild-type human alpha-2AAR gene identified as SEQID NO: 24 (GenBank Accession #AF281308 which includes the sequeniccorrections illuminated by Guyer et al, 1990) was amplified byoverlapping PCR reactions from genomic DNA derived from blood samples.The 1350 bp coding sequence as well as 341 bp 5′UTR and 174 bp 3′UTRwere examined. For convenience, the adenine of the initiator ATG codonis designated as nucleotide 1 and amino acid 1 is the encodedmethionine. The human receptor consists of 450 amino acids. For initialexamination, DNA from 27 hypertensive individuals was utilized.Overlapping PCR products encompassing the α_(2A) gene were designatedfragments 1-5 and were generated using the following primers: Fragment 1(600 bp), 5′-TTTACCCATCGGCTCTCCCTAC-3′ (sense) SEQ ID NO: 28 and5′-GAGACACCAGGAAGAGGTTTTGG-3′ (antisense) SEQ ID NO: 29; Fragment 2 (467bp) 5′-TCGTCATCATCGCCGTGTTC-3′ (sense) SEQ ID NO: 30 and5′-CGTACCACTTCTGGTCGTTGATC-3′ (antisense) SEQ ID NO: 31; Fragment 3 (556bp), 5′-GCCATCATCATCACCGTGTGGGTC-3′ (sense) SEQ ID NO: 32 and5′-GGCTCGCTCGGGCCTTGCCTTTG-3′ (antisense) SEQ ID NO: 33; Fragment 4 (436bp), 5′-GACCTGGAGGAGAGCTCGTCTT-3′ (sense) SEQ ID NO: 34 and5′-TGACCGGGTTCAACGAGCTGTTG-3′ (antisense) SEQ ID NO: 35; and Fragment 5(353 bp), 5′-GCCACGCACGCTCTTCAAATTCT-3′ (sense) SEQ ID NO: 36 and5′-TTCCCTTGTAGGAGCAGCAGAC-3′ (antisense) SEQ ID NO: 37. The 5′ end ofeach sense and antisense primer also contained sequence corresponding tothe M13 Forward (5′-TGTAAAACGACGGCCAGT) SEQ ID NO: 38 and M13 Reverse(5′-CAGGAAACAGCTATGACC) SEQ ID NO: 39 universal sequencing primers,respectively. The PCR consisted of ˜100 ng genomic DNA, 5 pmol of eachM13 primer, 0.8 mM dNTPs, 10% DMSO, 2.5 units Platinum taq DNApolymerase (Gibco/BRL), 20 uL 5×buffer A (Invitrogen) in a 100 μlreaction volume. Reactions were started by an initial incubation at 94°C. for four minutes, followed by 35 cycles of 94° C. for 30 seconds,denaturation for 30 seconds, and 72° C. for one minute, followed by afinal extension at 72° C. for seven minutes. The denaturationtemperature was 56° C. for fragments 1 and 5, 58° C. for fragments 2 and4, and 60° C. for fragment 3. PCR reactions were purified using QIAquickPCR purification system (Qiagen), and automated sequencing of bothstrands of each PCR product was performed using an Applied Biosystemssequencer using dye primer methods. As discussed, a C to G transversionat nucleotide 753 was identified that resulted in an asparagine tolysine change at amino acid 251 (shown in FIG. 5). This nucleotidechange results in gain of a unique Sty I restriction endonuclease sitein PCR fragment 3, and the presence or absence of this polymorphism inadditional samples was studied by Sty I digestion of fragment 3 PCRproducts (shown in FIG. 5, Panel D). This rapid detection technique wasapplied to additional DNA samples providing genotypes at this locus froma total of 376 individuals (normotensive: 125 Caucasian and 99African-American; hypertensive: 75 and 77 respectively). Normotensiveand hypertensive patients were selected as described previously by(14).

Example 8

[0290] Constructs and Cell Transfection

[0291] To create the polymorphic alpha-2AAR Lys251 construct, a portionof the coding region of alpha-2AAR gene containing a G at nucleotideposition 753 was amplified from a homozygous individual using fragment 2sense and fragment 4 antisense primers (see PCR conditions described inExample 7). This fragment was digested with and subcloned into the BglII and Sac II sites of the wild type α_(2A)AR sequence in the expressionvector pBC12B1. Chinese hamster ovary cells (CHO-K1) were permanentlytransfected by a calcium phosphate precipitation technique as previouslydescribed using 30 μg of each receptor construct and 3.0 μg of pSV₂neoto provide for G418 resistance by the methods of (15). Selection ofpositive clones was carried out in 1.0 mg/ml G418 and expression of thealpha-2AAR from individual clonal lines was determined by radioligandbinding as described below. Cells were grown in monolayers in Ham's F-12medium supplemented with 10% fetal calf serum, 100 units/ml penicillin,100 μg/ml streptomycin and 80 μg/ml G418 (to maintain selectionpressure) at 37° C. in a 5% CO₂ atmosphere. COS-7 cells wereco-transfected with 1-10 μg of each alpha-2AAR construct and ˜5 μgG_(iα) using a DEAE-dextran method essentially as described previouslyby (16). These transfections also included 5 μg of the large T antigencontaining plasmid, pRSVT (17), to maximize expression of the α_(2A)ARgene from the SV40 promoter of pBC12B1.

Example 9

[0292] Adenylyl Cyclase Activities

[0293] Alpha-2AAR inhibition of adenylyl cyclase was determined inmembrane preparations from CHO cells stably expressing the two receptorsusing methods similar to those previously described (18). Reactionsconsisted of 20 μg cell membranes, 2.7 mM phosphoenolpyruvate, 50 μMGTP, 0.1 mM cAMP, 0.12 mM ATP, 50 μg/ml myokinase, 0.05 mM ascorbic acidand 2 μCi of [α-³²P]ATP in a buffer containing 40 mM HEPES, pH 7.4, 1.6mM MgCl₂ and 0.8 mM EDTA for 30 minutes at 37° C. Reactions wereterminated by the addition of a stop solution containing excess ATP andcAMP and ˜100,000 dpm of [³H]cAMP. Labeled cAMP was isolated by gravitychromatography over alumina columns with [³H]cAMP used to quantitatecolumn recovery. Activities were measured in the presence of water(basal), 5 μM forskolin, and 5 μM forskolin with the indicatedconcentrations of agonists. Results are expressed as percent inhibitionof forskolin stimulated activity.

Example 10

[0294] [³⁵S]GTPγS Binding

[0295] Receptor-G protein interaction was quantitated by [³⁵S]radiolabeled guanosine-5′-O-(3-thiotriphosphate) ([³⁵S]GTPγS) binding inCOS-7 cells transiently transfected with each alpha-2AAR construct andG_(iα2). Briefly, cell membranes (˜20 μg) were incubated in buffercontaining 25 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 1 mM EDTA, 1 mMdithiotheritol, 100 mM NaCl, 1 μM GDP, and 2 nM [³⁵S]GTPγS in a 100 μlreaction volume for 15 min at room temperature. Incubations wereterminated by dilution with 4 volumes of ice cold 10 mM Tris-HCL, pH 7.4buffer and vacuum filtration over Whatmann GF/C glass fiber filters.Nonspecific binding was measured in the presence of 10 μM GTPγS.

Example 11

[0296] MAP Kinase Activation

[0297] Activation of p44/42 MAP kinase was determined by quantitativeimmunoblotting using specific antibodies to identify phosphorylated andtotal MAP kinase expression. Briefly, confluent cells were incubatedovernight in serum-free media prior to treatment with media alone(basal), epinephrine (10 μM), or thrombin (1 unit/ml) for 5 min. Cellswere washed three times with phosphate-buffered saline (PBS) then lysedin RIPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40,0.5% deoxycholate, 0.1% SDS, and 5 mM NaF) containing proteaseinhibitors (10 μg/ml benzamidine, 10 μg/ml soybean trypsin inhibitor, 10μg/ml aprotinin, and 5 μg/ml leupeptin). Western blots of these wholecell lysates were performed essentially as previously described (19).Membranes were incubated with phospho-p44/42 MAP kinase E10 antibody(New England Biolabs, Beverly, Mass.) at a dilution of 1:2000 for 1 hrat room temperature. Washed membranes were subsequently incubated withanti-mouse fluorescein-linked immunoglobulin followed by incubation withfluorescein alkaline phosphatase (ECF, Amersham). Fluorescent signalswere quantitated by real-time acquisition using a Molecular DynamicsSTORM imager. After stripping, membranes were incubated under the sameconditions as described in Example 4 with a p44/42 MAP kinase monoclonalantibody to quantitate total MAP kinase expression.

Example 12

[0298] Inositol Phosphate Accumulation

[0299] Total inositol phosphate levels in intact cells were determinedessentially as described previously (20). Briefly, confluent CHO cellsstably expressing each of the alpha-2ARRs were incubated with[³H]myoinositol (5 μCi/ml) in media lacking fetal calf serum for 16 hrsat 37° C. in 5% CO₂ atmosphere. Subsequently, cells were washed andincubated with PBS for 30 min followed by a 30 min incubation with 20 mMLiCl in PBS. Cells were then treated with PBS alone (basal), varyingconcentrations of epinephrine, or 5 units/ml thrombin for 5 min, andinositol phosphates were extracted as described by Martin (21).Following separation on Agl-X8 columns, total inositol phosphates wereeluted with a solution containing 0.1 M formic acid and 1 M formate.

Example 13

[0300] Radioligand Binding

[0301] Expression of mutant and wild-type alpha-2AAR was determinedusing saturation binding assays as described (22) with 12 concentrations(0.5-30 nM) of [³H]yohimbine and 10 μM phentolamine used to definenonspecific binding. For competition studies, membranes were incubatedin 50 mM Tris-HCL, pH 7.4, 10 mM MgSO₄, 0.5 mM EDTA with 2.0 nM[³H]yohimbine and 16 concentrations of the indicated competitor in thepresence of 100 μM GppNHp for 30 minutes at 37° C. Reactions for theabove radioligand binding studies were terminated by dilution with 4volumes of ice cold 10 mM Tris-HCL, pH 7.4 buffer and vacuum filtrationover Whatmann GF/C glass fiber filters.

Example 14

[0302] Protein Determination and Data Correlation

[0303] Protein determinations were by the copper bicinchoninic acidmethod (23). Data from adenylyl cyclase and radioligand binding assayswere analyzed by iterative least-square techniques using Prizm software(GraphPad, San Diego, Calif.). Agreement between genotypes observed andthose predicted by the Hardy-Weinberg equilibrium was assessed by aChi-squared test with one degree of freedom. Genotype comparisons wereby Fisher's exact test. Comparisons of results from biochemical studieswere paired by t-tests and significance was considered when p<0.05. Dataare provided as means±standard errors.

Results and Discussion of Examples 7-14

[0304] Sequence analysis of the entire coding region of the alpha-2AARgene from 54 chromosomes revealed one nonsynonymous sequence variantlocated within the third intracellular loop of the receptor (FIG. 5).This consisted of a C to G transversion at nucleotide 753 that changedamino acid 251 from Asn to Lys (FIG. 6). While the Lys251 receptor isrelatively rare, it is ˜10 fold more common in African-Americans than inCaucasians, with an allele frequency of 0.05 as compared to 0.004(p=0.01). The distribution of homozygous and heterozygous alleles wasnot different than that predicted from Hardy-Weinberg equilibrium(p>0.9). Two previously unreported synonymous single nucleotidepolymorphisms were also identified at nucleic acids 849 (C to G) and1093 (C to A). Considering the role of the alpha-2AAR in regulatingblood pressure, we also determined the frequency of this polymorphism inpatients with essential hypertension. Our analysis of 99 normotensiveand 77 hypertensive African-Americans as well as 125 normotensive and 75hypertensive Caucasians showed no differences in the frequency of thispolymorphism in patients with essential hypertension in either group.

[0305] The consequences of this polymorphism on ligand binding andreceptor function were evaluated by permanently expressing the humanwild-type alpha-2AAR and the Lys251 polymorphic receptor in CHO cells.Saturation radioligand binding studies revealed essentially identicaldissociation binding constants for the alpha-2AAR antagonist[³H]yohimbine (K_(d)=3.4±0.21 vs 3.6±0.25 nM respectively, n=4), andcompetition binding assays showed no differences in binding of theagonist (−) epinephrine (K_(i)=593±65 vs 734±31 nM respectively, n=3,Table 4). These data show that the ligand binding pocket composed of thetransmembrane spanning domains is not perturbed by the presence of Lysat amino acid 251 in the third intracellular loop. The Lys251polymorphism occurs in a highly conserved portion of the thirdintracellular loop of the α_(2A)AR (FIG. 6), in a region thought to beimportant for G-protein interaction (24). Indeed, as shown in FIG. 6,Asn is present in the position analogous to human 251 in all mammalianalpha-2AARs reported to date.

[0306] To assess whether this polymorphism affects G-protein coupling,functional studies examining agonist-promoted inhibition offorskolin-stimulated adenylyl cyclase activities were carried out incell lines expressing the wild type Asn251 receptor and the polymorphicLys251 receptor at levels of 2360±263 and 2590±140 fmol/mg (n=5,p>0.05), respectively. TABLE 4 Pharmacological Properties of the Asn251and Lys251 α_(2A)ARs expressed in CHO cells. Adenylyl RadioligandBinding cyclase activity ³H-Yo- Epineph- β_(max) himbine rine K₁ BasalForskolin Receptor (fmol/mg) K_(D) (nM) (nM) (pmol/min/mg) Asn251 2360 ±263 3.4 ± 0.21 593 ± 65 11.9 ± 2.3 32.7 ± 4.0 Lys251 2590 ± 140 3.6 ±0.25 734 ± 31 13.9 ± 1.6 31.6 ± 6.5

[0307] As shown in Table 4, basal and 5.0 μM forskolin-stimulatedadenylyl cyclase activities were not different between Asn251 and Lys251expressing cell lines, indicating that non-agonist dependent function isequivalent with the two receptors. However, activation of thepolymorphic Lys251 receptor with the full agonist epinephrine resultedin enhanced inhibition of adenylyl cyclase activity compared towild-type alpha-2AARs. Maximal inhibition of adenylyl cyclase was60±4.4% with the variant receptor compared to 46±4.1% with wild-type(n=5, p<0.005, FIG. 7a). Similar results were also found when receptorswere activated by the partial agonist oxymetazoline, with the Lys251having an ˜40% augmented function compared to the Asn251 receptor(50±6.6% vs 35±4.7% inhibition, n=5, p<0.05, FIG. 7b). No significantdifferences in the EC₅₀ values for epinephrine (583±196 nM vs 462±145nM) or for oxymetazoline (54.0±7.3 nM vs 67.3±15.7 nM) were observed.

[0308] This enhanced function was also found by quantitatingagonist-promoted receptor-G_(i) interaction with [³⁵S]GTPγS binding. Inthese experiments, Asn251 and Lys251 receptors were transientlycoexpressed in COS-7 cells (2.3±0.3 vs 2.2±1.4 pmol/mg) along withG_(iα2), and binding of [³⁵S]GTPγS was measured in membranes exposed tovehicle (basal) or saturating concentrations of various agonists. Here,full and partial agonists with diverse structures were utilized. As isshown in FIG. 8, the Lys251 receptor had increased [³⁵S]GTPγS binding inresponse to all agonists tested, albeit to varying degrees. Basal[³⁵S]GTPγS binding was equivalent. Stimulation with the full agonists UK14304, epinephrine, and norepinephrine resulted in ˜40% enhanced GTPγSbinding for the Lys251 receptor as compared to the Asn251 receptor. Onthe other hand, partial agonists displayed from 45% (BHT-933) up to 72%(guanabenz) enhancement of [³⁵S]GTPγS binding with the Lys251 receptor.These results are consistent with the adenylyl cyclase activity studieswhich also showed enhanced function of the polymorphic receptor. Inaddition, they indicate that the gain-of-function phenotype is morepronounced with some, but not all, partial agonists compared to fullagonists.

[0309] We next investigated agonist-mediated modulation of MAP kinase bywild-type and the Lys251 receptor. Alpha-2AAR act to stimulate MAPkinase activity and thus can potentially regulate cell growth anddifferentiation (2). While noting that regulation of MAP kinase activityis both receptor and cell-type specific, MAP kinase activation byalpha-2A receptors appears to be initiated by βγ released from G_(i)(2).

[0310] To investigate the extent of MAP kinase activation in CHO celllines expressing both the Asn251 and Lys251 receptors, quantitativeimmunoblots using an antibody specific to the activated (phosphorylated)form of ERK 1/2 were performed. While the total amount of MAP kinase wasnot different (FIG. 9A), agonist-promoted stimulation of MAP kinaseactivity was markedly different between the two cell lines (FIG. 9A, B).Activation of the Lys251 receptor with 10 μM epinephrine resulted in a57±9 fold increase in MAP kinase activity over basal as compared to15±2.1 fold increase with the Asn251 receptor (n=3, p=<0.05).

[0311] Finally, coupling of these two receptors to the stimulation ofinositol phosphate production was examined. Such α₂AR signaling is acomplex response due to activation of phospholipase C by βγ releasedfrom activated G_(o) and G_(i) (25). In contrast to the [³⁵S]GTPγSbinding, adenylyl cyclase, and MAP kinase results, the maximal extent ofepinephrine-stimulated accumulation of inositol phosphates was not foundto be different between Lys251 and Asn251 expressing cells (FIG. 6).However, the signal transduction of the LYS251 receptor was neverthelessenhanced, as evidenced by a decrease in the EC₅₀ (wildtype=493 mm,Lys251=119 mm, P<0.05).

[0312] Alpha-2AARs are widely expressed throughout the nervous systemand peripheral tissues. Recent work with relatively selective agonistsand antagonists, radiolabels, and specific molecular probes in severalspecies, including genetically engineered mice, have begun to elucidatespecific functions for the various alpha-2AAR subtypes (26). The latterstudies have been particularly useful in identifying subtype-specificfunctions. Mice lacking alpha-2AARs have higher resting systolic bloodpressures and more rapidly develop hypertension with sodium loadingafter subtotal nephrectomy than wild-type mice (27). Furthermore, thesealpha-2AAR knock-out mice fail to display a hypotensive response to theagonist dexmedetomidine (5). Heart rates in these mice were increased atrest, which correlated with increased [³H]norepinephrine release fromcardiac sympathetic nerves. These data thus indicate that thepresynaptic inhibition of neurotransmitter release in cortical andcardiac nerves serves important homeostatic functions in blood pressureand cardiac function. And, that the physiologic effects of therapeuticagonists such as clonidine reduce blood pressure by specifically actingat the alpha-2AAR subtype. The lack of a hypotensive effect ofalpha-2AAR agonists has also been shown in genetically altered(hit-and-run) mice expressing a dysfunctional alpha-2AAR (D79N) (28).These mice also responded poorly to alpha-2AAR agonists for severalother physiologic functions (6). Dexmedetomidine failed to reducerotarod latency or induce prolongation of sleep time, to enhance theefficacy of halothone, or to attenuate thermally induced pain in thesemice. Thus the sedative, anesthetic-sparing, and analgesic effects ofalpha-2AAR agonists are due to activation of the alpha-2AAR subtype.Indeed, these physiologic defects correlated with absent α_(2A)ARregulation of inwardly rectifying K⁺ channels of locus ceruleus neuronsand voltage gated Ca²⁺ channels of these same neurons, as well as thoseof the superior cervical ganglion (6).

[0313] Examples 7-14 indicate that a polymorphism resulting in amarkedly depressed alpha-2AAR function in humans would likely be ofphysiologic importance. Indeed, such a polymorphism can be a significantrisk factor for hypertension. However, the one coding block polymorphismthat we found in Caucasians and African-Americans is not associated withessential hypertension and in fact its phenotype is a gain of function.It should be noted that with our sample size we have the power to detecta polymorphism with an allele frequency of 0.04 with a statisticalcertainty of 90%, thus it is unlikely that we have failed to detectanother polymorphism that is common in any of the cohorts. Based on thephenotype of the Lys251 receptor, and the known physiologic function ofthe alpha-2AAR, one can predict that the polymorphism would predisposeto autonomic dysfunction characterized by hypotension and bradycardia.Similarly, patients with essential hypertension who have thepolymorphism can have milder disease or display enhanced efficacy ofantihypertensive agents such as clonidine or guanabenz. Interestingly,these individuals may display more pronounced central nervous systemside-effects from these agents, such as sedation, which could ultimatelylimit their therapeutic utility. Finally, the hyperfunctioningpolymorphism would be predicted to result in less norepinephrine releasefrom cardiac sympathetic nerves, thereby potentially providingprotection against the deleterious effects of catecholamines in patientswith heart failure.

[0314] Mutations of G-protein coupled receptors are the basis of anumber of rare diseases (29). In contrast, polymorphisms (allelefrequencies >1%) of these receptors have been identified which can beminor risk factors for complex diseases (30), but more importantly actas disease modifiers (31) or alter response to therapeutic agentstargeting the receptor (32). Interestingly, when such mutations orpolymorphisms have been found to alter function, the minor allelicvariant (i.e., the least common form of the receptor) results in eitherdecreased agonist-promoted function or increased non-agonist dependentfunction (i.e., constitutive activation). An example of the former isthe Ile164 polymorphisms of the β₂AR, which imparts defectiveagonist-promoted coupling to G_(s) (33). Constitutive activation resultsin receptors adopting a mutation induced agonist-bound like state andthus signaling becomes independent of agonist. Such persistentactivation is the pathologic basis for diseases such as male precociouspuberty, which is due to a mutation in the leutinizing hormone receptor(34). In the current report we show that the Lys251 receptor does notexhibit constitutive activation, based on wild-type [³S]GTPγS binding,adenylyl cyclase, and MAP kinase activities in the absence of agonist.Instead, the phenotype that we observed was one of increasedagonist-promoted function. To our knowledge this is the firstdelineation of a polymorphism of a pharmacogenetic locus of anyG-protein coupled receptor where the minor allele displays thisproperty, and thus this represents a new class of polymorphism for thesuperfamily.

[0315] Examples 15-21 below demonstrate that a polymorphism of thealpha-2AR subtype localized to human chromosome 4 (the pharmacologicalpha-2CAR subtype) within an intracellular domain has been identifiedin normal individuals. The polymorphism with amino acid deletions inpositions 322-325 of the alpha-2CAR (denoted Del322-325) is due to anin-frame 12 nucleic acid deletion encoding a receptor lackingGly-Ala-Gly-Pro in the third intracellular loop. Agonist binding studiesshowed decreased high affinity binding to the polymorphic receptor. Todelineate the functional consequences of this structural alteration,Chinese hamster ovary cells were permanently transfected with constructsencoding wild-type human alpha-2CAR and the polymorphic receptor. TheDel322-325 variant displayed markedly depressed epinephrine-promotedcoupling to G_(i), inhibiting adenylyl cyclase by 10±4.3% compared to73±2.4% for wild-type alpha-2CAR. This also was so for the endogenousligand norepinephrine and full and partial synthetic agonists. Depressedagonist-promoted coupling to the stimulation of MAP kinase (˜71%impaired) and inositol phosphate production (˜60% impaired) was alsofound with the polymorphic receptor. The Del322-325 receptor was ˜10times more frequent in African-Americans compared to Caucasians (allelefrequencies 0.381 vs 0.040). Given this significant loss-of-functionphenotype in several signal transduction cascades and the skewed ethnicprevalence, Del322-325 represents a pharmacoethnogenetic locus and canbe the basis for interindividual variation in diseases, such ascardiovascular or CNS pathophysiology.

Example 15

[0316] Polymorphism Detection

[0317] The nucleotide sequence encoding the third intracellular loop ofthe human alpha-2C adrenergic receptor (SEQ ID NO: 40) was examined forpolymorphic variation by performing polymerase chain reactions (PCR) toamplify this portion of the cDNA from genomic DNA derived from bloodsamples. For convenience, the adenine of the initiator ATG codon isdesignated as nucleotide 1 and amino acid 1 is the encoded methionine.The human receptor consists of 462 amino acids. For initial examination,DNA from 20 normal individuals was utilized. Primers for PCR were: SEQID NO:48 5′-CCACCATCGTCGCCGTGTGGCTCATCT-3′ (sense) and SEQ ID NO:495′-AGGCCTCGCGGCAGATGCCGTACA-3′ (antisense).

[0318] The PCR consisted of ˜100 ng genomic DNA, 5 pmol of each M13primer, 0.8 mM dNTPs, 10% DMSO, 2.5 units Platinum taq DNA polymeraseHigh Fidelity (Gibco/BRL), 20 uL 5×buffer E (Invitrogen) in a 100 μlreaction volume. Reactions were started by an initial incubation at 94°C. for four minutes, followed by 35 cycles of 94° C. for 30 seconds, 65°C. for 30 seconds, and 72° C. for one minute, followed by a finalextension at 72° C. for seven minutes. Attempts to directly sequencethis product resulted in ambiguous reads, so the product was ligatedinto the vector PCR2.1-TOPO (Invitrogen) and TOP 10 cells weretransformed. Multiple colonies from each transformation were expandedand the subsequently isolated DNA was sequenced using an ABI 373Aautomated sequencer in the forward and reverse directions using dyeterminator chemistry, such as dideoxy nucleotides, with vector T7 andM13 primers. As is discussed, a 12 bp deletion was found in someindividuals beginning at nucleotide 964 of FIG. 11. This results in theloss of amino acids 322-325 and thus this polymorphic receptor isdenoted Del 322-325. This deletion results in the loss of a Nci Irestriction site at nucleotide 974 (forward strand), and thus a rapiddetection method was devised. Smaller (384 and 372 base-pair) PCRproducts were produced using 5′-AGCCCGACGAGAGCAGCGCA-3′ SEQ ID NO: 50 asthe sense primer and the aforementioned antisense primer (same reactionconditions as above) and genomic DNA derived from blood samples as thetemplate. Within this fragment there are either five or six Ncirestriction sites depending on the presence or absence of the deletion,providing for the pattern shown in FIG. 11C. This rapid detectiontechnique was applied to additional DNA samples providing genotypes atthis locus from a total of 146 individuals. No other nonsynonymouspolymorphisms were found in the third intracellular loop sequence.However, five synonymous single nucleotide polymorphisms were found atnucleic acids 868, 871, 933, 996 and 1167.

Example 16

[0319] Constructs and Cell Transfection

[0320] To create the polymorphic alpha-2CAR construct the larger (723bp) PCR product described above amplified from a homozygous individualwas digested and subcloned into the Bpu1102 I and Eco47 III sites of thewild-type alpha-2CAR sequence in the expression vector pBC12BI (Eason etal. J.Biol.Chem. 267, 25473-25479 (1992)). The integrity of theconstruct was verified by sequencing. Chinese hamster ovary cells(CHO-K1) were permanently transfected by a calcium phosphateprecipitation technique as previously described using 30 μg of eachreceptor construct and 0.5 μg of pSV₂neo to provide for G418 resistance( Eason et al. J.Biol.Chem. 267, 25473-25479 (1992)). Selection ofpositive clones was carried out in 1.0 mg/ml G418 and expression of thealpha-2C receptors from individual clonal lines was determined byradioligand binding as described below. Cells were grown in monolayersin Ham's F-12 medium supplemented with 10% fetal calf serum, 100units/ml penicillin, 100 μg/ml streptomycin and 80 μg/ml G418 (tomaintain selection pressure) at 37° C. in a 5% CO₂ atmosphere.

Example 17

[0321] Adenylyl Cyclase Activity

[0322] Alpha-2AR inhibition of adenylyl cyclase was determined inmembrane preparation from CHO cells stably expressing the two receptorsusing methods similar to those previously described (Eason et al.J.Biol. Chem. 267, 25473-25479 (1992)). Briefly, membranes (˜20 μg) wereincubated with 27 μM phosphoenolpyruvate, 0.5 μM GTP, 0.1 mM cAMP, 0.12mM ATP, 50 μg/ml myokinase, 0.05 mM ascorbic acid and 2 μCi of[alpha-³²P]ATP in a buffer containing 40 mM HEPES, pH 7.4, 1.6 mM MgCl₂and 0.8 mM EDTA for 30 minutes at 37° C. These conditions minimize thestimulation of adenylyl cyclase which is observed at high agonistconcentrations (Fraser et al. J.Biol.Chem. 264 , 11754-11761 (1989);Eason et al. J.Biol.Chem. 267, 15795-15801 (1992)). Reactions wereterminated by the addition of a stop solution containing excess ATP andcAMP and ˜100,000 dpm of [³H]cAMP. Labeled cAMP was isolated by gravitychromatography over alumina columns with [³H]cAMP used to quantitatecolumn recovery. Activities were measured in the presence of water(basal), 5 μM forskolin, and 5 μM forskolin with the indicatedconcentrations of agonists. Results are expressed as percent inhibitionof forskolin stimulated activity.

Example 18

[0323] MAP Kinase Activation

[0324] Activation of p44/42 MAP kinase was determined by quantitativeimmunoblotting using a phospho-specific antibody. Briefly, confluentcells were incubated overnight at 37° C. and 5% CO₂ in serum-free mediaprior to treatment with media alone (basal), epinephrine (10 μM), orthrombin (1 unit/ml) for 5 min. Cells were washed three times withphosphate-buffered saline (PBS) then lysed in RIPA buffer (20 mM Tris,pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate, 0.1% SDS,and 5 mM NaF) containing protease inhibitors (10 μg/ml benzamidine, 10μg/ml soybean trypsin inhibitor, 10 μg/ml aprotinin, and 5 μg/mlleupeptin). Western blots of these whole cell lysates were performedessentially as previously described (18) except that PVDF membranes(Amersham) were used and incubated with phospho-p44/42 MAP kinase E10antibody, and (after stripping) with the p44/42 MAP kinase monoclonalantibody (both from New England Biolabs, Beverly, Mass.) at dilutions of1:2000 for 1 hr at room temperature. Washed membranes were subsequentlyincubated with anti-mouse fluorescein-linked immunoglobulin followed byincubation with fluorescein alkaline phosphatase (ECF, Amersham).Fluorescent signals were quantitated by real-time acquisition using aMolecular Dynamics STORM imager.

Example 19

[0325] Inositol Phosphate Accumulation

[0326] Total inositol phosphate levels in intact cells were determinedessentially as described previously (Schwinn et al. Mol.Pharmacol. 40,619-626 (1991)). Briefly, confluent CHO cells stably expressing each ofthe α_(2C)ARs were incubated with [³H]myoinositol (5 μCi/ml) in medialacking fetal calf serum for 16 hrs at 37° C. in 5% CO₂ atmosphere.Subsequently, cells were washed and incubated with PBS for 30 minfollowed by a 30 min incubation with 20 mM LiCl in PBS. Cells were thentreated with PBS alone (basal), 10 μM epinephrine, or 5 units/mlthrombin for 5 min, and inositol phosphates were extracted as describedby Martin (Martin, T. F. J. J Biol Chem 258, 14816-14822 (1983)).Following separation on Agl-X8 columns, total inositol phosphates wereeluted with a solution containing 0.1 M formic acid and 1 M formate.

Example 20

[0327] Radioligand Binding

[0328] Expression of mutant and wild-type alpha-2CAR was determinedusing saturation binding assays as described (Eason et al.Proc.Natl.Acad.Sci., USA 91, 11178-11182 (1994)) with 12 concentrations(0.5-30 nM) of [³H]yohimbine and 10 μM phentolamine used to definenonspecific binding. For competition studies, membranes were incubatedin 50 mM Tris-HCL, pH 7.4, 10 mM MgSO₄, 0.5 mM EDTA with 2.0 nM[³H]yohimbine and 16 concentrations of the indicated competitor for 30minutes at 37° C. Reactions for the above radioligand binding studieswere terminated by dilution with 4 volumes of ice cold 10 mM Tris-HCL,pH 7.4 buffer and vacuum filtration over Whatmann GF/C glass fiberfilters.

Example 21

[0329] Protein Determination and Data Correlation

[0330] Protein determinations were by the copper bicinchoninic acidmethod (Smith et al. Anal.Biochem. 150, 76-85 (1985)). Data fromadenylyl cyclase and radioligand binding assays were analyzed byiterative least-square techniques using Prizm software (GraphPad, SanDiego, Calif.). Agreement between genotypes observed and those predictedby the Hardy-Weinberg equilibrium was assessed by a Chi-squared testwith one degree of freedom. Comparisons of results from biochemicalstudies were paired by t-tests and significance was considered whenp<0.05. Data are provided as means standard errors.

Results and Discussion of Examples 15-21

[0331] From the initial sequencing of alpha-2CAR third intracellularloop PCR products from 40 chromosomes, one nonsynonymous sequencevariant was identified (FIG. 11). This consisted of an in-frame 12nucleotide (SEQ ID NO: 41 ggggcggggccg, sense strand) deletion beginningat nucleotide 964 of FIG. 11. This results in a loss of Gly-Ala-Gly-Proat amino acid positions 322-325 within the third intracellular loop ofthe receptor (FIG. 12) SEQ ID NO: 45. The frequencies of the wild-typeand the Del322-325 polymorphic alpha-2CARs are shown in Table 5. Thepolymorphism is rare in Caucasians with an allelic frequency of 0.040.In contrast, the frequency is ˜10-fold higher (0.381) inAfrican-Americans. The distribution of homozygous and heterozygousalleles was not different than that predicted from the Hardy-Weinbergequilibrium (p>0.8). TABLE 5 Frequencies of the Del322-325 alpha-2CARpolymorphism Allele WT Heterozy- Del 322-325 Frequency Del N Homozygousgous Homozygous 322-325 Caucasian 87 82 3 2 0.040 African- 59 23 27 90.381 American

[0332] The consequences of this polymorphism on receptor function wereevaluated by permanently expressing the human wild-type alpha-2CAR andthe Del322-325 receptor in CHO cells and examining multiple signalingpathways. As indicated, multiple clones with similar expression levelswere utilized for these studies. Saturation radioligand binding studiesusing the alpha-2AR antagonist [³H]yohimbine revealed that Del322-325had a slightly, but statistically significant, lower affinity for theradioligand compared to wild-type alpha-2CAR (K_(d)=3.8 0.55 vs 2.0 0.14nM, n=5, p=0.03). In competition studies with the agonist epinephrine,carried out in the absence of GTP, high- and low-affinity binding wasdetected with both receptors. However the high affinity dissociationconstant K_(H) for the Del322-325 mutant was greater (i.e., loweraffinity) compared to the wild-type receptor (7.3 0.95 vs 3.7 0.43 nM,n=4, p=0.01. And, the percentage of receptors in the high-affinity statewas less with the mutant receptor (% R_(H)32 31 4 vs 49 4, p=0.01). TheK_(L) values were not different (584 71 vs 416 75 nM). Taken together,this indicates impaired formation of the high affinityagonist-receptor-G_(i)/G_(o) complex. TABLE 6 Adenylyl cyclaseactivities of the wild-type and Del322-325 alpha-2CAR for full andpartial agonists. Max Inhibition (%)* EC₅₀ (nM) Del322- AgonistWTα_(2C)AR 325 WTα_(2C)AR Del322-325 Norepinephrine 76.6 ± 1.60 34.9 ± 219 ± 13.7  224 ± 70.7 0.887 UK 14304 67.6 ± 2.09 30.8 ±  131 ± 31.0 109 ± 24.1 4.68 BHT-933 56.8 ± 1.41 26.7 ± 5500 ± 2110 4080 ± 2005 2.40guanabenz 53.8 ± 1.99 30.0 ± 1.98 clonidine 38.5 ± 2.44 20.9 ±  262 ±26.1  178 ± 43.8 1.28 oxymetazoline 27.0 ± 2.70 12.8 ± 32.2 ± 1.66  29.1± 0.565 1.40

[0333] The location of the deletion in the third intracellular loop ofthe receptor is within 15 residues of the sequence RRGGRR SEQ ID NO: 51.This is a motif that has been identified in a number of receptors as aG_(i) coupling domain (Okamoto et al. J.Biol. Chem. 267, 8342-8346(1992); Ikezu et al. FEBS 311, 29-32 (1992)). The deletion of the twoglycines or the proline in the Del322-325 receptor inducesconformational changes affecting this region or other G-protein couplingdomains. Functional studies examining agonist-promoted inhibition offorskolin stimulated adenylyl cyclase activities were carried out inlines with the wild-type alpha-2CAR and the Del322-325 receptor atexpression levels of 1375±141 vs 1081±157 fmol/mg (n=5, p>0.05) and asecond set of lines with lower expressions of 565±69 vs 519±51 fmol/mg(n=5, p>0.05), respectively. The results of these studies are shown inFIG. 13. As can be seen, there is a marked functional difference betweenthe two receptors. In the higher expressing lines (FIG. 13A), wild-typealpha-2CAR exhibited a maximal inhibitory response of 60±3%. Incontrast, the Del322-325 polymorphic receptor achieved a maximalinhibition of 31±2% (n=5, p<0.001), which represents an ˜50% impairmentof function. Of note, the EC₅₀s for these responses (2.6±0.74 vs1.2±0.37 nM, respectively) were not different.

[0334] Results from studies with the lower expressing lines revealed aneven more striking phenotypic difference between the two receptors. Asis shown in FIG. 13B, at these more physiologic levels of expression,agonist-promoted inhibition of adenylyl cyclase with wild-typealpha-2CAR was 73±2.4%. In marked contrast, the Del322-325 receptorexhibited very little inhibition (10±4.3%, n=5, p<0.001). With the lowexpressing Del322-325 line the EC₅₀ in some experiments could not becalculated due to the minimal response. Analysis of the composite curveof the mean data from the above examples with this line revealed an EC₅₀of 29.6 nM. This is in contrast to 4.3 nM calculated in a like mannerfor the low expressing wild-type line. A similar degree of impairmentwas also observed with the endogenous agonist norepinephrine (Table 6).Agonist-promoted functional activities of the two higher expressingreceptors were also explored with full and partial synthetic alpha-2ARagonists with diverse structures. As is shown in Table 6, the Del322-325receptor has depressed agonist-promoted coupling to inhibition ofadenylyl cyclase with all the agonists tested.

[0335] We next explored coupling of these two receptors (mutant andwild-type) to the stimulation of inositol phosphate production. In CHOcells this response is ablated by pertussis toxin, indicating couplingvia G_(i) and/or G_(o) (Dorn et al. Biochem 36, 6415-6423 (1997)). Theactivation of phopholipase C is likely due to both G_(o)-alpha mediatedstimulation and G_(i) associated G beta gamma stimulation of the enzyme(Dom et al. Biochem 36, 6415-6423 (1997)). As shown in FIG. 14, theloss-of-function phenotype of the Del322-325 receptor as delineated inadenylyl cyclase experiments was also observed in these inositolphosphate accumulation studies. Epinephrine-stimulated accumulation ofinositol phosphates was 30±3% over basal with the wild-type alpha-2CAR,compared to 11±2% for the Del322-325 receptor (n=4, p<0.005) whichamounts to an ˜60% impairment of function for the polymorphic receptor.Expression levels for the two receptors for these experiments were806±140 and 733±113 respectively.

[0336] Agonist mediated stimulation of MAP kinase was examined. Themechanism of G protein coupled receptor mediated stimulation of thispathway is multifactorial and is both receptor and cell-type dependent(Luttrell et al. Adv Second Messenger Phosphoprotein Res 31, 263-277(1997)). For the beta2AR, coupling to G_(i), internalization of thereceptor, and interaction with beta-arrestin is required for thisreceptor to activate the MAP kinase cascade. Alpha-2AR coupling to thispathway is pertussis toxin sensitive and receptor internalization is notnecessary (Schramm et al. J Biol Chem 274, 24935-24940 (1999)). Forpresent studies, MAP kinase activation was assessed using quantitativeimmunoblots with an antibody specific for the activated (phosphorylated)form of Erk1/2. The total amount of MAP kinase was not different betweenthe two cell lines utilized (FIG. 15A). Agonist promoted activation ofMAP kinase was significantly different between the two receptors (FIG.15), with results expressed both as the agonist-promoted fold increaseover basal levels of activated MAP kinase and as the percent of thethrombin response. In five such experiments, MAPK activity in Del322-325expressing cells in response to 10 μM epinephrine was 57.8±7.0% of theWT alpha-2CAR response (p<0.005), and the stimulation as a percent ofthe thrombin response was 128±10.0 vs 37.2±5.7 (p<0.005), respectively.

[0337] Recent studies have begun to elucidate specific functions for thealpha-2CAR subtype. In situ mRNA and immunohistochemical analysis ofalpha-2CAR expression has revealed a distinct pattern of expression inrat brain and spinal cord (Rosin et al. J Comp Neurol 372, 135-165(1996); Shi et al. Neuroreport 10, 2835-2839 (1999)). Alpha-2CARs havebeen localized primarily in the neuronal perikarya and to a lesserextent in the proximal dendrites, with high levels of receptorexpression detected in the basal ganglia, olfactory tubercle,hippocampus, and cerebral cortex (Rosin et al. J Comp Neurol 372,135-165 (1996)). These data along with studies of genetically engineeredmice indicate that the alpha-2CAR subtype plays explicit roles incognitive and behavioral functions. Studies of mice that overexpress, orthat have targeted inactivation of, the alpha-2CAR gene have shown thatthis receptor is involved in the regulation of spontaneous motoractivity as well as agonist-induced regulation of body temperature anddopamine metabolism (Sallinen et al. Mol.Pharmacol. 51, 36-46 (1997)).

[0338] In addition, results indicating that activation of alpha-2CARreduces hyperreactivity and impulsivity have also been reported(Sallinen et al. The Journal of Neuroscience 18, 3035-3042 (1998)).These studies show that lack of alpha-2CAR expression is associated withincreased startle reactivity, reduced prepulse inhibition of the startlereflex, and isolation induced attack latency, while overexpression ofalpha-2CAR produces the opposite effects. Consistent with these data, inhumans, the alpha-2AR agonist clonidine and the alpha-2AR antagonistidazoxan reduce and facilitate the acoustic startle response,respectively (Morgan et al. Psychopharmacology 110, 342-346 (1993);Kumari et al. Psychopharmacology 123, 353-360 (1996)). The role ofalpha-2CAR in modulating working memory has also been characterized(Tanila et al. European Journal of Neuroscience 11, 599-603 (1999)). Inthese studies, alpha-2CAR knockout mice performed less accurately in adelayed alternation task and displayed slowed motor initiation in thereturn phase of the task, supporting a role for the alpha-2CAR in thecognitive aspect of response preparation. Alpha-2CAR knockout mice werealso impaired in spatial and non-spatial water maze tests, thussupporting a role for this receptor in modulating cognitive functions(Bjorklund et al. Mol Pharmacol 54, 569-576 (1998)), and alteration ofalpha-2CAR expression in transgenic mice has also been linked withbehavioral despair development and changes in plasma corticosteronelevels (Sallinen et al. Mol Psychiatry 4, 443-452 (1999)).

[0339] Recent studies measuring [³H]norepinephrine release from centralneurons and cardiac sympathetic nerves have shown that thefrequency-release curves for alpha-2CAR deficient mice are rightwardshifted compared to wild-type mice (Hein et al. Nature 402, 181-184(1999)). Furthermore, the residual agonist-stimulated inhibition of[³H]norepinephrine release observed in alpha-2AAR deficient mice was notpresent in mice deficient in both alpha-2A and alpha-2CAR. Thus bothsubtypes are important in inhibiting neurotransmitter release at thesesites. Alpha-2CAR mRNA or receptor protein has also been identified inother peripheral sites ( Gavin et al. Naunyn Schmiedebergs ArchPharmacol 355, 406-411 (1997); Eason et al. Mol.Pharmacol. 44, 70-75(1993); Adolfsson et al. Gynecol Obstet Invest 45, 145-150 (1998)) withevidence in some cases indicative of postsynaptic functions (Gavin etal. Naunyn Schmiedebergs Arch Pharmacol 355, 406-411 (1997)).

[0340] The presence of functionally distinct polymorphic alpha-2CARsaccounts for interindividual variability in physiological responses, andis the basis of differences in clinical characteristics of diseaseswhere alpha-2CAR function is important. In addition, the Del322-325polymorphism can predispose individuals to the development of disease.The results show that response to agonist or antagonist therapeuticagents may also vary depending on receptor genotype. In this regardindividuals with Del322-325 are more sensitive to antagonists since theyhave receptors which are less responsive to endogenous catecholamines.For agonists, the response or sensitivity would be predicted to be lessfor those with the polymorphic alpha-2CAR due to its impaired coupling.The results discussed in Table 5 show relatively high frequency of thepolymorphism in healthy African-Americans, and modification of a diseaseor drug-response. We and others have recently shown that functionalpolymorphisms of the α₂AR indeed appear to have one or more of the aboveeffects in asthma, congestive heart failure, and obesity (Tan et al.Lancet 350, 995-999 (1997); Tan et al. Lancet 350, 995-999 (1997); Largeet al. J Clin Invest 100, 3005-3013 (1997)). Interestingly, Comings etal (Comings et al. Clin Genet 55, 160-172 (1999)) have found thatincreased levels of plasma norepinephrine levels in children withattention-deficit hyperactivity disorder (ADHD) with learningdisabilities were associated with polymorphisms near the coding regionsof the alpha-2A, alpha-2C, and dopamine β-hydroxylase (DBH) genes.

[0341] In summary, examples 15-21 demonstrate that a polymorphicalpha-2CAR has been identified that includes a deletion of four aminoacids in the third intracellular loop of the receptor. Such a deletionhas a significant effect on agonist-promoted inhibition of adenylylcyclase, stimulation of inositol phosphate accumulation and activationof MAP kinase. For all three effector pathways, the Del322-325 receptordisplays markedly impaired coupling. The polymorphism is rare inCaucasians, but is ˜10 fold more prevalent in African-Americans with anallele frequency of 0.381. To our knowledge, this is the greatest racialdifference in a polymorphism of any G-protein coupled receptor reportedto date. Given the extreme phenotype, this locus is considered a basisfor interindividual variation in physiologic responses, diseasepredisposition, or modification, and drug responsiveness.

[0342] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice thin theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims. TABLE 7 Frequency of the Alpha-2BAR Del301-303polymorphism. Del301-303 Wt Homo- Del301-303 Allele n zygousHeterozygous Homozygous Frequency Caucasian 94 41 47 6 0.31 African- 7961 17 1 0.12 American

[0343] TABLE 8 Ligand binding properties of wild-type and Del301-303alpha-2BAR expressed in CHO cells [³H]-yohimbine [¹²⁵I]-aminoclonidinesaturation binding epinephrine competition saturation binding ReceptorB_(max) (fmol/mg) K_(D) (nM) K_(i) K_(H) (nM) K_(L) (nM) % R_(H) B_(max)(fmol/mg) K_(D) (nM) Wild-Type 671 ± 56 3.8 ± 0.3 285 ± 8.7  2.9 ± 0.8346 ± 111 41 ± 4 118 ± 27 1.33 ± 0.12 Del301-303 538 ± 79  5.1 ± 0.2*376 ± 66* 4.1 ± 1.2 357 ± 135 42 ± 5 106 ± 20 1.22 ± .07 

[0344] TABLE 9 Adenylyl cyclase activities of the wild-type andDel301-303 alpha-2BAR expressed in CHO cells Max Basal ForskolinInhibition Submax inhibition (%) Desensitization pmol/min/mg (%) EC₅₀(nM) Ctrl NE (%) Wild Type 2.0 ± 0.2  15.1 ± 0.9  28.5 ± 1.6  7.9 ± 2.116.5 ± 3.9  7.6 ± 2.3^(†) 54 Del301-303 1.2 ± 0.1* 11.9 ± 0.9* 23.4 ±2.2* 19.6 ± 5.5* 17.1 ± 3.0 15.9 ± 1.7* 7

[0345]

1 53 1 1353 DNA Homo sapiens 1 atggaccacc aggaccccta ctccgtgcaggccacagcgg ccatagcggc ggccatcacc 60 ttcctcattc tctttaccat cttcggcaacgctctggtca tcctggctgt gttgaccagc 120 cgctcgctgc gcgcccctca gaacctgttcctggtgtcgc tggccgccgc cgacatcctg 180 gtggccacgc tcatcatccc tttctcgctggccaacgagc tgctgggcta ctggtacttc 240 cggcgcacgt ggtgcgaggt gtacctggcgctcgacgtgc tcttctgcac ctcgtccatc 300 gtgcacctgt gcgccatcag cctggaccgctactgggccg tgagccgcgc gctggagtac 360 aactccaagc gcaccccgcg ccgcatcaagtgcatcatcc tcactgtgtg gctcatcgcc 420 gccgtcatct cgctgccgcc cctcatctacaagggcgacc agggccccca gccgcgcggg 480 cgcccccagt gcaagctcaa ccaggaggcctggtacatcc tggcctccag catcggatct 540 ttctttgctc cttgcctcat catgatccttgtctacctgc gcatctacct gatcgccaaa 600 cgcagcaacc gcagaggtcc cagggccaagggggggcctg ggcagggtga gtccaagcag 660 ccccgacccg accatggtgg ggctttggcctcagccaaac tgccagccct ggcctctgtg 720 gcttctgcca gagaggtcaa cggacactcgaagtccactg gggagaagga ggagggggag 780 acccctgaag atactgggac ccgggccttgccacccagtt gggctgccct tcccaactca 840 ggccagggcc agaaggaggg tgtttgtggggcatctccag aggatgaagc tgaagaggag 900 gaagaggagg aggaggagga ggaagagtgtgaaccccagg cagtgccagt gtctccggcc 960 tcagcttgca gccccccgct gcagcagccacagggctccc gggtgctggc caccctacgt 1020 ggccaggtgc tcctgggcag gggcgtgggtgctataggtg ggcagtggtg gcgtcgaagg 1080 gcgcagctga cccgggagaa gcgcttcaccttcgtgctgg ctgtggtcat tggcgttttt 1140 gtgctctgct ggttcccctt cttcttcagctacagcctgg gcgccatctg cccgaagcac 1200 tgcaaggtgc cccatggcct cttccagttcttcttctgga tcggctactg caacagctca 1260 ctgaaccctg ttatctacac catcttcaaccaggacttcc gccgtgcctt ccggaggatc 1320 ctgtgccgcc cgtggaccca gacggcctggtga 1353 2 1344 DNA Homo sapiens 2 atggaccacc aggaccccta ctccgtgcaggccacagcgg ccatagcggc ggccatcacc 60 ttcctcattc tctttaccat cttcggcaacgctctggtca tcctggctgt gttgaccagc 120 cgctcgctgc gcgcccctca gaacctgttcctggtgtcgc tggccgccgc cgacatcctg 180 gtggccacgc tcatcatccc tttctcgctggccaacgagc tgctgggcta ctggtacttc 240 cggcgcacgt ggtgcgaggt gtacctggcgctcgacgtgc tcttctgcac ctcgtccatc 300 gtgcacctgt gcgccatcag cctggaccgctactgggccg tgagccgcgc gctggagtac 360 aactccaagc gcaccccgcg ccgcatcaagtgcatcatcc tcactgtgtg gctcatcgcc 420 gccgtcatct cgctgccgcc cctcatctacaagggcgacc agggccccca gccgcgcggg 480 cgcccccagt gcaagctcaa ccaggaggcctggtacatcc tggcctccag catcggatct 540 ttctttgctc cttgcctcat catgatccttgtctacctgc gcatctacct gatcgccaaa 600 cgcagcaacc gcagaggtcc cagggccaagggggggcctg ggcagggtga gtccaagcag 660 ccccgacccg accatggtgg ggctttggcctcagccaaac tgccagccct ggcctctgtg 720 gcttctgcca gagaggtcaa cggacactcgaagtccactg gggagaagga ggagggggag 780 acccctgaag atactgggac ccgggccttgccacccagtt gggctgccct tcccaactca 840 ggccagggcc agaaggaggg tgtttgtggggcatctccag aggatgaagc tgaagaggag 900 gaggaggagg aggaagagtg tgaaccccaggcagtgccag tgtctccggc ctcagcttgc 960 agccccccgc tgcagcagcc acagggctcccgggtgctgg ccaccctacg tggccaggtg 1020 ctcctgggca ggggcgtggg tgctataggtgggcagtggt ggcgtcgaag ggcgcagctg 1080 acccgggaga agcgcttcac cttcgtgctggctgtggtca ttggcgtttt tgtgctctgc 1140 tggttcccct tcttcttcag ctacagcctgggcgccatct gcccgaagca ctgcaaggtg 1200 ccccatggcc tcttccagtt cttcttctggatcggctact gcaacagctc actgaaccct 1260 gttatctaca ccatcttcaa ccaggacttccgccgtgcct tccggaggat cctgtgccgc 1320 ccgtggaccc agacggcctg gtga 1344 39 DNA Homo sapiens 3 gaagaggag 9 4 9 DNA Homo sapiens 4 gaggaggag 9 5 9DNA Homo sapiens 5 cttctcctc 9 6 9 DNA Homo sapiens 6 ctcctcctc 9 7 450PRT Homo sapiens 7 Met Asp His Gln Asp Pro Tyr Ser Val Gln Ala Thr AlaAla Ile Ala 1 5 10 15 Ala Ala Ile Thr Phe Leu Ile Leu Phe Thr Ile PheGly Asn Ala Leu 20 25 30 Val Ile Leu Ala Val Leu Thr Ser Arg Ser Leu ArgAla Pro Gln Asn 35 40 45 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp Ile LeuVal Ala Thr Leu 50 55 60 Ile Ile Pro Phe Ser Leu Ala Asn Glu Leu Leu GlyTyr Trp Tyr Phe 65 70 75 80 Arg Arg Thr Trp Cys Glu Val Tyr Leu Ala LeuAsp Val Leu Phe Cys 85 90 95 Thr Ser Ser Ile Val His Leu Cys Ala Ile SerLeu Asp Arg Tyr Trp 100 105 110 Ala Val Ser Arg Ala Leu Glu Tyr Asn SerLys Arg Thr Pro Arg Arg 115 120 125 Ile Lys Cys Ile Ile Leu Thr Val TrpLeu Ile Ala Ala Val Ile Ser 130 135 140 Leu Pro Pro Leu Ile Tyr Lys GlyAsp Gln Gly Pro Gln Pro Arg Gly 145 150 155 160 Arg Pro Gln Cys Lys LeuAsn Gln Glu Ala Trp Tyr Ile Leu Ala Ser 165 170 175 Ser Ile Gly Ser PhePhe Ala Pro Cys Leu Ile Met Ile Leu Val Tyr 180 185 190 Leu Arg Ile TyrLeu Ile Ala Lys Arg Ser Asn Arg Arg Gly Pro Arg 195 200 205 Ala Lys GlyGly Pro Gly Gln Gly Glu Ser Lys Gln Pro Arg Pro Asp 210 215 220 His GlyGly Ala Leu Ala Ser Ala Lys Leu Pro Ala Leu Ala Ser Val 225 230 235 240Ala Ser Ala Arg Glu Val Asn Gly His Ser Lys Ser Thr Gly Glu Lys 245 250255 Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro 260265 270 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu Gly Val275 280 285 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu Glu Glu GluGlu 290 295 300 Glu Glu Glu Glu Glu Cys Glu Pro Gln Ala Val Pro Val SerPro Ala 305 310 315 320 Ser Ala Cys Ser Pro Pro Leu Gln Gln Pro Gln GlySer Arg Val Leu 325 330 335 Ala Thr Leu Arg Gly Gln Val Leu Leu Gly ArgGly Val Gly Ala Ile 340 345 350 Gly Gly Gln Trp Trp Arg Arg Arg Ala GlnLeu Thr Arg Glu Lys Arg 355 360 365 Phe Thr Phe Val Leu Ala Val Val IleGly Val Phe Val Leu Cys Trp 370 375 380 Phe Pro Phe Phe Phe Ser Tyr SerLeu Gly Ala Ile Cys Pro Lys His 385 390 395 400 Cys Lys Val Pro His GlyLeu Phe Gln Phe Phe Phe Trp Ile Gly Tyr 405 410 415 Cys Asn Ser Ser LeuAsn Pro Val Ile Tyr Thr Ile Phe Asn Gln Asp 420 425 430 Phe Arg Arg AlaPhe Arg Arg Ile Leu Cys Arg Pro Trp Thr Gln Thr 435 440 445 Ala Trp 4508 447 PRT Homo sapiens 8 Met Asp His Gln Asp Pro Tyr Ser Val Gln Ala ThrAla Ala Ile Ala 1 5 10 15 Ala Ala Ile Thr Phe Leu Ile Leu Phe Thr IlePhe Gly Asn Ala Leu 20 25 30 Val Ile Leu Ala Val Leu Thr Ser Arg Ser LeuArg Ala Pro Gln Asn 35 40 45 Leu Phe Leu Val Ser Leu Ala Ala Ala Asp IleLeu Val Ala Thr Leu 50 55 60 Ile Ile Pro Phe Ser Leu Ala Asn Glu Leu LeuGly Tyr Trp Tyr Phe 65 70 75 80 Arg Arg Thr Trp Cys Glu Val Tyr Leu AlaLeu Asp Val Leu Phe Cys 85 90 95 Thr Ser Ser Ile Val His Leu Cys Ala IleSer Leu Asp Arg Tyr Trp 100 105 110 Ala Val Ser Arg Ala Leu Glu Tyr AsnSer Lys Arg Thr Pro Arg Arg 115 120 125 Ile Lys Cys Ile Ile Leu Thr ValTrp Leu Ile Ala Ala Val Ile Ser 130 135 140 Leu Pro Pro Leu Ile Tyr LysGly Asp Gln Gly Pro Gln Pro Arg Gly 145 150 155 160 Arg Pro Gln Cys LysLeu Asn Gln Glu Ala Trp Tyr Ile Leu Ala Ser 165 170 175 Ser Ile Gly SerPhe Phe Ala Pro Cys Leu Ile Met Ile Leu Val Tyr 180 185 190 Leu Arg IleTyr Leu Ile Ala Lys Arg Ser Asn Arg Arg Gly Pro Arg 195 200 205 Ala LysGly Gly Pro Gly Gln Gly Glu Ser Lys Gln Pro Arg Pro Asp 210 215 220 HisGly Gly Ala Leu Ala Ser Ala Lys Leu Pro Ala Leu Ala Ser Val 225 230 235240 Ala Ser Ala Arg Glu Val Asn Gly His Ser Lys Ser Thr Gly Glu Lys 245250 255 Glu Glu Gly Glu Thr Pro Glu Asp Thr Gly Thr Arg Ala Leu Pro Pro260 265 270 Ser Trp Ala Ala Leu Pro Asn Ser Gly Gln Gly Gln Lys Glu GlyVal 275 280 285 Cys Gly Ala Ser Pro Glu Asp Glu Ala Glu Glu Glu Glu GluGlu Glu 290 295 300 Glu Glu Cys Glu Pro Gln Ala Val Pro Val Ser Pro AlaSer Ala Cys 305 310 315 320 Ser Pro Pro Leu Gln Gln Pro Gln Gly Ser ArgVal Leu Ala Thr Leu 325 330 335 Arg Gly Gln Val Leu Leu Gly Arg Gly ValGly Ala Ile Gly Gly Gln 340 345 350 Trp Trp Arg Arg Arg Ala Gln Leu ThrArg Glu Lys Arg Phe Thr Phe 355 360 365 Val Leu Ala Val Val Ile Gly ValPhe Val Leu Cys Trp Phe Pro Phe 370 375 380 Phe Phe Ser Tyr Ser Leu GlyAla Ile Cys Pro Lys His Cys Lys Val 385 390 395 400 Pro His Gly Leu PheGln Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser 405 410 415 Ser Leu Asn ProVal Ile Tyr Thr Ile Phe Asn Gln Asp Phe Arg Arg 420 425 430 Ala Phe ArgArg Ile Leu Cys Arg Pro Trp Thr Gln Thr Ala Trp 435 440 445 9 16 PRTHomo sapiens 9 Glu Asp Glu Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu GluGlu Glu 1 5 10 15 10 13 PRT Homo sapiens 10 Glu Asp Glu Ala Glu Glu GluGlu Glu Glu Glu Glu Glu 1 5 10 11 3 PRT Homo sapiens 11 Glu Glu Glu 1 123 PRT Homo sapiens 12 Cys Glu Pro 1 13 21 DNA Homo sapiens 13 gctcatcatccctttctcgc t 21 14 21 DNA Homo sapiens 14 aaagccccac catggtcggg t 21 1520 DNA Homo sapiens 15 ctgatcgcca aacgagcaac 20 16 20 DNA Homo sapiens16 aaaaacgcca atgaccacag 20 17 18 DNA Homo sapiens 17 tgtaaaacgacggccagt 18 18 18 DNA Homo sapiens 18 caggaaacag ctatgacc 18 19 21 DNAHomo sapiens 19 agaaggaggg tgtttgtggg g 21 20 21 DNA Homo sapiens 20acctatagca cccacgcccc t 21 21 21 DNA Homo sapiens 21 ggccgacgctcttgtctagc c 21 22 20 DNA Homo sapiens 22 caaggggttc ctaagatgag 20 23 9PRT Homo sapiens 23 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 24 1350 DNAHomo sapiens 24 atgggctccc tgcagccgga cgcgggcaac gcgagctgga acgggaccgaggcgccgggg 60 ggcggcgccc gggccacccc ttactccctg caggtgacgc tgacgctggtgtgcctggcc 120 ggcctgctca tgctgctcac cgtgttcggc aacgtgctcg tcatcatcgccgtgttcacg 180 agccgcgcgc tcaaggcgcc ccaaaacctc ttcctggtgt ctctggcctcggccgacatc 240 ctggtggcca cgctcgtcat ccctttctcg ctggccaacg aggtcatgggctactggtac 300 ttcggcaagg cttggtgcga gatctacctg gcgctcgacg tgctcttctgcacgtcgtcc 360 atcgtgcacc tgtgcgccat cagcctggac cgctactggt ccatcacacaggccatcgag 420 tacaacctga agcgcacgcc gcgccgcatc aaggccatca tcatcaccgtgtgggtcatc 480 tcggccgtca tctccttccc gccgctcatc tccatcgaga agaagggcggcggcggcggc 540 ccgcagccgg ccgagccgcg ctgcgagatc aacgaccaga agtggtacgtcatctcgtcg 600 tgcatcggct ccttcttcgc tccctgcctc atcatgatcc tggtctacgtgcgcatctac 660 cagatcgcca agcgtcgcac ccgcgtgcca cccagccgcc ggggtccggacgccgtcgcc 720 gcgccgccgg ggggcaccga gcgcaggccc aacggtctgg gccccgagcgcagcgcgggc 780 ccggggggcg cagaggccga accgctgccc acccagctca acggcgcccctggcgagccc 840 gcgccggccg ggccgcgcga caccgacgcg ctggacctgg aggagagctcgtcttccgac 900 cacgccgagc ggcctccagg gccccgcaga cccgagcgcg gtccccggggcaaaggcaag 960 gcccgagcga gccaggtgaa gccgggcgac agcctgccgc ggcgcgggccgggggcgacg 1020 gggatcggga cgccggctgc agggccgggg gaggagcgcg tcggggctgccaaggcgtcg 1080 cgctggcgcg ggcggcagaa ccgcgagaag cgcttcacgt tcgtgctggccgtggtcatc 1140 ggagtgttcg tggtgtgctg gttccccttc ttcttcacct acacgctcacggccgtcggg 1200 tgctccgtgc cacgcacgct cttcaaattc ttcttctggt tcggctactgcaacagctcg 1260 ttgaacccgg tcatctacac catcttcaac cacgatttcc gccgcgccttcaagaagatc 1320 ctctgtcggg gggacaggaa gcggatcgtg 1350 25 1350 DNA Homosapiens 25 atgggctccc tgcagccgga cgcgggcaac gcgagctgga acgggaccgaggcgccgggg 60 ggcggcgccc gggccacccc ttactccctg caggtgacgc tgacgctggtgtgcctggcc 120 ggcctgctca tgctgctcac cgtgttcggc aacgtgctcg tcatcatcgccgtgttcacg 180 agccgcgcgc tcaaggcgcc ccaaaacctc ttcctggtgt ctctggcctcggccgacatc 240 ctggtggcca cgctcgtcat ccctttctcg ctggccaacg aggtcatgggctactggtac 300 ttcggcaagg cttggtgcga gatctacctg gcgctcgacg tgctcttctgcacgtcgtcc 360 atcgtgcacc tgtgcgccat cagcctggac cgctactggt ccatcacacaggccatcgag 420 tacaacctga agcgcacgcc gcgccgcatc aaggccatca tcatcaccgtgtgggtcatc 480 tcggccgtca tctccttccc gccgctcatc tccatcgaga agaagggcggcggcggcggc 540 ccgcagccgg ccgagccgcg ctgcgagatc aacgaccaga agtggtacgtcatctcgtcg 600 tgcatcggct ccttcttcgc tccctgcctc atcatgatcc tggtctacgtgcgcatctac 660 cagatcgcca agcgtcgcac ccgcgtgcca cccagccgcc ggggtccggacgccgtcgcc 720 gcgccgccgg ggggcaccga gcgcaggccc aagggtctgg gccccgagcgcagcgcgggc 780 ccggggggcg cagaggccga accgctgccc acccagctca acggcgcccctggcgagccc 840 gcgccggccg ggccgcgcga caccgacgcg ctggacctgg aggagagctcgtcttccgac 900 cacgccgagc ggcctccagg gccccgcaga cccgagcgcg gtccccggggcaaaggcaag 960 gcccgagcga gccaggtgaa gccgggcgac agcctgccgc ggcgcgggccgggggcgacg 1020 gggatcggga cgccggctgc agggccgggg gaggagcgcg tcggggctgccaaggcgtcg 1080 cgctggcgcg ggcggcagaa ccgcgagaag cgcttcacgt tcgtgctggccgtggtcatc 1140 ggagtgttcg tggtgtgctg gttccccttc ttcttcacct acacgctcacggccgtcggg 1200 tgctccgtgc cacgcacgct cttcaaattc ttcttctggt tcggctactgcaacagctcg 1260 ttgaacccgg tcatctacac catcttcaac cacgatttcc gccgcgccttcaagaagatc 1320 ctctgtcggg gggacaggaa gcggatcgtg 1350 26 450 PRT Homosapiens 26 Met Gly Ser Leu Gln Pro Asp Ala Gly Asn Ala Ser Trp Asn GlyThr 1 5 10 15 Glu Ala Pro Gly Gly Gly Ala Arg Ala Thr Pro Tyr Ser LeuGln Val 20 25 30 Thr Leu Thr Leu Val Cys Leu Ala Gly Leu Leu Met Leu LeuThr Val 35 40 45 Phe Gly Asn Val Leu Val Ile Ile Ala Val Phe Thr Ser ArgAla Leu 50 55 60 Lys Ala Pro Gln Asn Leu Phe Leu Val Ser Leu Ala Ser AlaAsp Ile 65 70 75 80 Leu Val Ala Thr Leu Val Ile Pro Phe Ser Leu Ala AsnGlu Val Met 85 90 95 Gly Tyr Trp Tyr Phe Gly Lys Ala Trp Cys Glu Ile TyrLeu Ala Leu 100 105 110 Asp Val Leu Phe Cys Thr Ser Ser Ile Val His LeuCys Ala Ile Ser 115 120 125 Leu Asp Arg Tyr Trp Ser Ile Thr Gln Ala IleGlu Tyr Asn Leu Lys 130 135 140 Arg Thr Pro Arg Arg Ile Lys Ala Ile IleIle Thr Val Trp Val Ile 145 150 155 160 Ser Ala Val Ile Ser Phe Pro ProLeu Ile Ser Ile Glu Lys Lys Gly 165 170 175 Gly Gly Gly Gly Pro Gln ProAla Glu Pro Arg Cys Glu Ile Asn Asp 180 185 190 Gln Lys Trp Tyr Val IleSer Ser Cys Ile Gly Ser Phe Phe Ala Pro 195 200 205 Cys Leu Ile Met IleLeu Val Tyr Val Arg Ile Tyr Gln Ile Ala Lys 210 215 220 Arg Arg Thr ArgVal Pro Pro Ser Arg Arg Gly Pro Asp Ala Val Ala 225 230 235 240 Ala ProPro Gly Gly Thr Glu Arg Arg Pro Asn Gly Leu Gly Pro Glu 245 250 255 ArgSer Ala Gly Pro Gly Gly Ala Glu Ala Glu Pro Leu Pro Thr Gln 260 265 270Leu Asn Gly Ala Pro Gly Glu Pro Ala Pro Ala Gly Pro Arg Asp Thr 275 280285 Asp Ala Leu Asp Leu Glu Glu Ser Ser Ser Ser Asp His Ala Glu Arg 290295 300 Pro Pro Gly Pro Arg Arg Pro Glu Arg Gly Pro Arg Gly Lys Gly Lys305 310 315 320 Ala Arg Ala Ser Gln Val Lys Pro Gly Asp Ser Leu Pro ArgArg Gly 325 330 335 Pro Gly Ala Thr Gly Ile Gly Thr Pro Ala Ala Gly ProGly Glu Glu 340 345 350 Arg Val Gly Ala Ala Lys Ala Ser Arg Trp Arg GlyArg Gln Asn Arg 355 360 365 Glu Lys Arg Phe Thr Phe Val Leu Ala Val ValIle Gly Val Phe Val 370 375 380 Val Cys Trp Phe Pro Phe Phe Phe Thr TyrThr Leu Thr Ala Val Gly 385 390 395 400 Cys Ser Val Pro Arg Thr Leu PheLys Phe Phe Phe Trp Phe Gly Tyr 405 410 415 Cys Asn Ser Ser Leu Asn ProVal Ile Tyr Thr Ile Phe Asn His Asp 420 425 430 Phe Arg Arg Ala Phe LysLys Ile Leu Cys Arg Gly Asp Arg Lys Arg 435 440 445 Ile Val 450 27 450PRT Homo sapiens 27 Met Gly Ser Leu Gln Pro Asp Ala Gly Asn Ala Ser TrpAsn Gly Thr 1 5 10 15 Glu Ala Pro Gly Gly Gly Ala Arg Ala Thr Pro TyrSer Leu Gln Val 20 25 30 Thr Leu Thr Leu Val Cys Leu Ala Gly Leu Leu MetLeu Leu Thr Val 35 40 45 Phe Gly Asn Val Leu Val Ile Ile Ala Val Phe ThrSer Arg Ala Leu 50 55 60 Lys Ala Pro Gln Asn Leu Phe Leu Val Ser Leu AlaSer Ala Asp Ile 65 70 75 80 Leu Val Ala Thr Leu Val Ile Pro Phe Ser LeuAla Asn Glu Val Met 85 90 95 Gly Tyr Trp Tyr Phe Gly Lys Ala Trp Cys GluIle Tyr Leu Ala Leu 100 105 110 Asp Val Leu Phe Cys Thr Ser Ser Ile ValHis Leu Cys Ala Ile Ser 115 120 125 Leu Asp Arg Tyr Trp Ser Ile Thr GlnAla Ile Glu Tyr Asn Leu Lys 130 135 140 Arg Thr Pro Arg Arg Ile Lys AlaIle Ile Ile Thr Val Trp Val Ile 145 150 155 160 Ser Ala Val Ile Ser PhePro Pro Leu Ile Ser Ile Glu Lys Lys Gly 165 170 175 Gly Gly Gly Gly ProGln Pro Ala Glu Pro Arg Cys Glu Ile Asn Asp 180 185 190 Gln Lys Trp TyrVal Ile Ser Ser Cys Ile Gly Ser Phe Phe Ala Pro 195 200 205 Cys Leu IleMet Ile Leu Val Tyr Val Arg Ile Tyr Gln Ile Ala Lys 210 215 220 Arg ArgThr Arg Val Pro Pro Ser Arg Arg Gly Pro Asp Ala Val Ala 225 230 235 240Ala Pro Pro Gly Gly Thr Glu Arg Arg Pro Lys Gly Leu Gly Pro Glu 245 250255 Arg Ser Ala Gly Pro Gly Gly Ala Glu Ala Glu Pro Leu Pro Thr Gln 260265 270 Leu Asn Gly Ala Pro Gly Glu Pro Ala Pro Ala Gly Pro Arg Asp Thr275 280 285 Asp Ala Leu Asp Leu Glu Glu Ser Ser Ser Ser Asp His Ala GluArg 290 295 300 Pro Pro Gly Pro Arg Arg Pro Glu Arg Gly Pro Arg Gly LysGly Lys 305 310 315 320 Ala Arg Ala Ser Gln Val Lys Pro Gly Asp Ser LeuPro Arg Arg Gly 325 330 335 Pro Gly Ala Thr Gly Ile Gly Thr Pro Ala AlaGly Pro Gly Glu Glu 340 345 350 Arg Val Gly Ala Ala Lys Ala Ser Arg TrpArg Gly Arg Gln Asn Arg 355 360 365 Glu Lys Arg Phe Thr Phe Val Leu AlaVal Val Ile Gly Val Phe Val 370 375 380 Val Cys Trp Phe Pro Phe Phe PheThr Tyr Thr Leu Thr Ala Val Gly 385 390 395 400 Cys Ser Val Pro Arg ThrLeu Phe Lys Phe Phe Phe Trp Phe Gly Tyr 405 410 415 Cys Asn Ser Ser LeuAsn Pro Val Ile Tyr Thr Ile Phe Asn His Asp 420 425 430 Phe Arg Arg AlaPhe Lys Lys Ile Leu Cys Arg Gly Asp Arg Lys Arg 435 440 445 Ile Val 45028 22 DNA Homo sapiens 28 tttacccatc ggctctccct ac 22 29 23 DNA Homosapiens 29 gagacaccag gaagaggttt tgg 23 30 20 DNA Homo sapiens 30tcgtcatcat cgccgtgttc 20 31 23 DNA Homo sapiens 31 cgtaccactt ctggtcgttgatc 23 32 24 DNA Homo sapiens 32 gccatcatca tcaccgtgtg ggtc 24 33 23 DNAHomo sapiens 33 ggctcgctcg ggccttgcct ttg 23 34 22 DNA Homo sapiens 34gacctggagg agagctcgtc tt 22 35 23 DNA Homo sapiens 35 tgaccgggttcaacgagctg ttg 23 36 23 DNA Homo sapiens 36 gccacgcacg ctcttcaaat tct 2337 22 DNA Homo sapiens 37 ttcccttgta ggagcagcag ac 22 38 18 DNA Homosapiens 38 tgtaaaacga cggccagt 18 39 18 DNA Homo sapiens 39 caggaaacagctatgacc 18 40 1386 DNA Homo sapiens 40 atggcgtccc cggcgctggc ggcggcgctggcggtggcgg cagcggcggg ccccaatgcg 60 agcggcgcgg gcgagagggg cagcggcggggttgccaatg cctcgggggc ttcctggggg 120 ccgccgcgcg gccagtactc ggcgggcgcggtggcagggc tggctgccgt ggtgggcttc 180 ctcatcgtct tcaccgtggt gggcaacgtgctggtggtga tcgccgtgct gaccagccgg 240 gcgctgcgcg cgccacagaa cctcttcctggtgtcgctgg cctcggccga catcctggtg 300 gccacgctgg tcatgccctt ctcgttggccaacgagctca tggcctactg gtacttcggg 360 caggtgtggt gcggcgtgta cctggcgctcgatgtgctgt tttgcacctc gtcgatcgtg 420 catctgtgtg ccatcagcct ggaccgctactggtcggtga cgcaggccgt cgagtacaac 480 ctgaagcgca caccacgccg cgtcaaggccaccatcgtcg ccgtgtggct catctcggcc 540 gtcatctcct tcccgccgct ggtctcgctctaccgccagc ccgacggcgc cgcctacccg 600 cagtgcggcc tcaacgacga gacctggtacatcctgtcct cctgcatcgg ctccttcttc 660 gcgccctgcc tcatcatggg cctggtctacgcgcgcatct accgagtggc caagcgtcgc 720 acgcgcacgc tcagcgagaa gcgcgcccccgtgggccccg acggtgcgtc cccgactacc 780 gaaaacgggc tgggcgcggc ggcaggcgcaggcgagaacg ggcactgcgc gcccccgccc 840 gccgacgtgg agccggacga gagcagcgcagcggccgaga ggcggcggcg ccggggcgcg 900 ttgcggcggg gcgggcggcg gcgagcgggcgcggaggggg gcgcgggcgg tgcggacggg 960 cagggggcgg ggccgggggc ggctgagtcgggggcgctga ccgcctccag gtccccgggg 1020 cccggtggcc gcctctcgcg cgccagctcgcgctccgtcg agttcttcct gtcgcgccgg 1080 cgccgggcgc gcagcagcgt gtgccgccgcaaggtggccc aggcgcgcga gaagcgcttc 1140 acctttgtgc tggctgtggt catgggcgtgttcgtgctct gctggttccc cttcttcttc 1200 atctacagcc tgtacggcat ctgccgcgaggcctgccagg tgcccggccc gctcttcaag 1260 ttcttcttct ggatcggcta ctgcaacagctcgctcaacc cggtcatcta cacggtcttc 1320 aaccaggatt tccggccatc cttcaagcacatcctcttcc gacggaggag aaggggcttc 1380 aggcag 1386 41 12 DNA Homo sapiens41 ggggcggggc cg 12 42 1374 DNA Homo sapiens 42 atggcgtccc cggcgctggcggcggcgctg gcggtggcgg cagcggcggg ccccaatgcg 60 agcggcgcgg gcgagaggggcagcggcggg gttgccaatg cctcgggggc ttcctggggg 120 ccgccgcgcg gccagtactcggcgggcgcg gtggcagggc tggctgccgt ggtgggcttc 180 ctcatcgtct tcaccgtggtgggcaacgtg ctggtggtga tcgccgtgct gaccagccgg 240 gcgctgcgcg cgccacagaacctcttcctg gtgtcgctgg cctcggccga catcctggtg 300 gccacgctgg tcatgcccttctcgttggcc aacgagctca tggcctactg gtacttcggg 360 caggtgtggt gcggcgtgtacctggcgctc gatgtgctgt tttgcacctc gtcgatcgtg 420 catctgtgtg ccatcagcctggaccgctac tggtcggtga cgcaggccgt cgagtacaac 480 ctgaagcgca caccacgccgcgtcaaggcc accatcgtcg ccgtgtggct catctcggcc 540 gtcatctcct tcccgccgctggtctcgctc taccgccagc ccgacggcgc cgcctacccg 600 cagtgcggcc tcaacgacgagacctggtac atcctgtcct cctgcatcgg ctccttcttc 660 gcgccctgcc tcatcatgggcctggtctac gcgcgcatct accgagtggc caagcgtcgc 720 acgcgcacgc tcagcgagaagcgcgccccc gtgggccccg acggtgcgtc cccgactacc 780 gaaaacgggc tgggcgcggcggcaggcgca ggcgagaacg ggcactgcgc gcccccgccc 840 gccgacgtgg agccggacgagagcagcgca gcggccgaga ggcggcggcg ccggggcgcg 900 ttgcggcggg gcgggcggcggcgagcgggc gcggaggggg gcgcgggcgg tgcggacggg 960 cagggggcgg ctgagtcgggggcgctgacc gcctccaggt ccccggggcc cggtggccgc 1020 ctctcgcgcg ccagctcgcgctccgtcgag ttcttcctgt cgcgccggcg ccgggcgcgc 1080 agcagcgtgt gccgccgcaaggtggcccag gcgcgcgaga agcgcttcac ctttgtgctg 1140 gctgtggtca tgggcgtgttcgtgctctgc tggttcccct tcttcttcat ctacagcctg 1200 tacggcatct gccgcgaggcctgccaggtg cccggcccgc tcttcaagtt cttcttctgg 1260 atcggctact gcaacagctcgctcaacccg gtcatctaca cggtcttcaa ccaggatttc 1320 cggccatcct tcaagcacatcctcttccga cggaggagaa ggggcttcag gcag 1374 43 12 DNA Homo sapiens 43ggggcggctg ag 12 44 462 PRT Homo sapiens 44 Met Ala Ser Pro Ala Leu AlaAla Ala Leu Ala Val Ala Ala Ala Ala 1 5 10 15 Gly Pro Asn Ala Ser GlyAla Gly Glu Arg Gly Ser Gly Gly Val Ala 20 25 30 Asn Ala Ser Gly Ala SerTrp Gly Pro Pro Arg Gly Gln Tyr Ser Ala 35 40 45 Gly Ala Val Ala Gly LeuAla Ala Val Val Gly Phe Leu Ile Val Phe 50 55 60 Thr Val Val Gly Asn ValLeu Val Val Ile Ala Val Leu Thr Ser Arg 65 70 75 80 Ala Leu Arg Ala ProGln Asn Leu Phe Leu Val Ser Leu Ala Ser Ala 85 90 95 Asp Ile Leu Val AlaThr Leu Val Met Pro Phe Ser Leu Ala Asn Glu 100 105 110 Leu Met Ala TyrTrp Tyr Phe Gly Gln Val Trp Cys Gly Val Tyr Leu 115 120 125 Ala Leu AspVal Leu Phe Cys Thr Ser Ser Ile Val His Leu Cys Ala 130 135 140 Ile SerLeu Asp Arg Tyr Trp Ser Val Thr Gln Ala Val Glu Tyr Asn 145 150 155 160Leu Lys Arg Thr Pro Arg Arg Val Lys Ala Thr Ile Val Ala Val Trp 165 170175 Leu Ile Ser Ala Val Ile Ser Phe Pro Pro Leu Val Ser Leu Tyr Arg 180185 190 Gln Pro Asp Gly Ala Ala Tyr Pro Gln Cys Gly Leu Asn Asp Glu Thr195 200 205 Trp Tyr Ile Leu Ser Ser Cys Ile Gly Ser Phe Phe Ala Pro CysLeu 210 215 220 Ile Met Gly Leu Val Tyr Ala Arg Ile Tyr Arg Val Ala LysArg Arg 225 230 235 240 Thr Arg Thr Leu Ser Glu Lys Arg Ala Pro Val GlyPro Asp Gly Ala 245 250 255 Ser Pro Thr Thr Glu Asn Gly Leu Gly Ala AlaAla Gly Ala Gly Glu 260 265 270 Asn Gly His Cys Ala Pro Pro Pro Ala AspVal Glu Pro Asp Glu Ser 275 280 285 Ser Ala Ala Ala Glu Arg Arg Arg ArgArg Gly Ala Leu Arg Arg Gly 290 295 300 Gly Arg Arg Arg Ala Gly Ala GluGly Gly Ala Gly Gly Ala Asp Gly 305 310 315 320 Gln Gly Ala Gly Pro GlyAla Ala Glu Ser Gly Ala Leu Thr Ala Ser 325 330 335 Arg Ser Pro Gly ProGly Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser 340 345 350 Val Glu Phe PheLeu Ser Arg Arg Arg Arg Ala Arg Ser Ser Val Cys 355 360 365 Arg Arg LysVal Ala Gln Ala Arg Glu Lys Arg Phe Thr Phe Val Leu 370 375 380 Ala ValVal Met Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe 385 390 395 400Ile Tyr Ser Leu Tyr Gly Ile Cys Arg Glu Ala Cys Gln Val Pro Gly 405 410415 Pro Leu Phe Lys Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu 420425 430 Asn Pro Val Ile Tyr Thr Val Phe Asn Gln Asp Phe Arg Pro Ser Phe435 440 445 Lys His Ile Leu Phe Arg Arg Arg Arg Arg Gly Phe Arg Gln 450455 460 45 4 PRT Homo sapiens 45 Gly Ala Gly Pro 1 46 458 PRT Homosapiens 46 Met Ala Ser Pro Ala Leu Ala Ala Ala Leu Ala Val Ala Ala AlaAla 1 5 10 15 Gly Pro Asn Ala Ser Gly Ala Gly Glu Arg Gly Ser Gly GlyVal Ala 20 25 30 Asn Ala Ser Gly Ala Ser Trp Gly Pro Pro Arg Gly Gln TyrSer Ala 35 40 45 Gly Ala Val Ala Gly Leu Ala Ala Val Val Gly Phe Leu IleVal Phe 50 55 60 Thr Val Val Gly Asn Val Leu Val Val Ile Ala Val Leu ThrSer Arg 65 70 75 80 Ala Leu Arg Ala Pro Gln Asn Leu Phe Leu Val Ser LeuAla Ser Ala 85 90 95 Asp Ile Leu Val Ala Thr Leu Val Met Pro Phe Ser LeuAla Asn Glu 100 105 110 Leu Met Ala Tyr Trp Tyr Phe Gly Gln Val Trp CysGly Val Tyr Leu 115 120 125 Ala Leu Asp Val Leu Phe Cys Thr Ser Ser IleVal His Leu Cys Ala 130 135 140 Ile Ser Leu Asp Arg Tyr Trp Ser Val ThrGln Ala Val Glu Tyr Asn 145 150 155 160 Leu Lys Arg Thr Pro Arg Arg ValLys Ala Thr Ile Val Ala Val Trp 165 170 175 Leu Ile Ser Ala Val Ile SerPhe Pro Pro Leu Val Ser Leu Tyr Arg 180 185 190 Gln Pro Asp Gly Ala AlaTyr Pro Gln Cys Gly Leu Asn Asp Glu Thr 195 200 205 Trp Tyr Ile Leu SerSer Cys Ile Gly Ser Phe Phe Ala Pro Cys Leu 210 215 220 Ile Met Gly LeuVal Tyr Ala Arg Ile Tyr Arg Val Ala Lys Arg Arg 225 230 235 240 Thr ArgThr Leu Ser Glu Lys Arg Ala Pro Val Gly Pro Asp Gly Ala 245 250 255 SerPro Thr Thr Glu Asn Gly Leu Gly Ala Ala Ala Gly Ala Gly Glu 260 265 270Asn Gly His Cys Ala Pro Pro Pro Ala Asp Val Glu Pro Asp Glu Ser 275 280285 Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Leu Arg Arg Gly 290295 300 Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly305 310 315 320 Gln Gly Ala Ala Glu Ser Gly Ala Leu Thr Ala Ser Arg SerPro Gly 325 330 335 Pro Gly Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser ValGlu Phe Phe 340 345 350 Leu Ser Arg Arg Arg Arg Ala Arg Ser Ser Val CysArg Arg Lys Val 355 360 365 Ala Gln Ala Arg Glu Lys Arg Phe Thr Phe ValLeu Ala Val Val Met 370 375 380 Gly Val Phe Val Leu Cys Trp Phe Pro PhePhe Phe Ile Tyr Ser Leu 385 390 395 400 Tyr Gly Ile Cys Arg Glu Ala CysGln Val Pro Gly Pro Leu Phe Lys 405 410 415 Phe Phe Phe Trp Ile Gly TyrCys Asn Ser Ser Leu Asn Pro Val Ile 420 425 430 Tyr Thr Val Phe Asn GlnAsp Phe Arg Pro Ser Phe Lys His Ile Leu 435 440 445 Phe Arg Arg Arg ArgArg Gly Phe Arg Gln 450 455 47 4 PRT Homo sapiens 47 Gly Ala Ala Glu 148 27 DNA Homo sapiens 48 ccaccatcgt cgccgtgtgg ctcatct 27 49 24 DNAHomo sapiens 49 aggcctcgcg gcagatgccg taca 24 50 20 DNA Homo sapiens 50agccggacga gagcagcgca 20 51 6 PRT Homo sapiens 51 Arg Arg Gly Gly ArgArg 1 5 52 12 DNA Homo sapiens 52 ccccgccccg gc 12 53 12 DNA Homosapiens 53 ccccgccgac tc 12

What is claimed is:
 1. An isolated polynucleotide encoding an alpha-2C adrenergic receptor molecule or fragment or complement of the polynucleotide, having a polymorphic site comprising ggggcggctgag (SEQ ID NO: 43) at nucleotide positions 964 to
 975. 2. An isolated polynucleotide according to claim 1, wherein the polynucleotide comprises ggggcggctgag (SEQ ID NO: 43) at nucleotide positions 964 to 975 with up to 20 additional nucleotides on either the 5′ or 3′ end or both.
 3. An isolated oligonucleotide that specifically hybridizes to a region of a polynucleotide encoding an alpha-2C adrenergic receptor molecule or fragment of the polynucleotide, wherein the region comprises at least one polymorphic site comprising ggggcggctgag (SEQ ID NO: 43) at nucleotide positions 964 to
 975. 4. An isolated oligonucleotide according to claim 3, wherein the oligonucleotide permits identification of one polymorphic site comprising ggggcggctgag (SEQ ID NO: 43) at nucleotide positions 964 to
 975. 5. An isolated oligonucleotide according to claim 3, wherein the oligonucleotide is a primer that specifically hybridizes to a region immediately adjacent to nucleotide positions 964 to
 975. 6. A method of genotyping an alpha-2C-adrenergic receptor gene comprising: (a) obtaining a sample having a polynucleotide encoding an alpha-2C-adrenergic receptor molecule or fragment or complement of the polynucleotide; and (b) detecting in the sample a polymorphic site comprising SEQ ID NO: 41 or SEQ ID NO: 43 at nucleotide positions 964 to 975 of the polynucleotide or fragment or complement of the polynucleotide.
 7. A method according to claim 6, wherein the polymorphic site is detected by terminator sequencing, restriction digestion, allele-specific polymerase reaction, single-stranded conformational polymorphism analysis, genetic bit analysis, temperature gradient gel electrophoresis, ligase chain reaction, or ligase/polymerase genetic bit analysis.
 8. A method according to claim 6, wherein the polymorphic site is detected by employing nucleotides with a detectable characteristic selected from the group consisting of inherent mass, electric charge, electron spin, mass tag, radioactive isotope, dye, bioluminescent molecule, chemiluminescent molecule, nucleic acid molecule, hapten molcule, protein molecule, light scattering/phase shifting molecule, or fluorescence molecule.
 9. A method of detecting a polymorphic site in a sample to determine alpha-2C-adrenergic receptor function, comprising: (a) obtaining the sample having a polynucleotide encoding an alpha-2C-adrenergic receptor molecule or fragment or complement of the polynucleotide; and (b) detecting in the sample a polymorphic site comprising SEQ ID NO: 41 or SEQ ID NO: 43 at nucleotide positions 964 to 975 of the polynucleotide or fragment or complement of the polynucleotide.
 10. A method according to claim 9, wherein the polymorphic site is detected by terminator sequencing, restriction digestion, allele-specific polymerase reaction, single-stranded conformational polymorphism analysis, genetic bit analysis, temperature gradient gel electrophoresis, ligase chain reaction, or ligase/polymerase genetic bit analysis.
 11. A method according to claim 9, wherein the polymorphic site is detected by employing nucleotides with a detectable characteristic selected from the group consisting of inherent mass, electric charge, electron spin, mass tag, radioactive isotope, dye, bioluminescent molecule, chemiluminescent molecule, nucleic acid molecule, hapten molcule, protein molecule, light scattering/phase shifting molecule, or fluorescence molecule.
 12. A method of predicting an individual's response to an agonist or antagonist, comprising: (a) obtaining a sample having a polynucleotide encoding an alpha-2C-adrenergic receptor molecule or fragment or complement of the polynucleotide from the individual; (b) detecting in the sample a polymorphic site comprising SEQ ID NO: 41 or SEQ ID NO: 43 at nucleotide positions 964 to 975 of the polynucleotide or fragment or complement of the polynucleotide; and (c) correlating the polymorphic site to a predetermined response thereby predicting the individual's response to the agonist or antagonist.
 13. A method according to claim 12, wherein the agonist is an alpha-2C adrenergic receptor agonist.
 14. A method according to claim 12, wherein the antagonist is an alpha-2C adrenergic receptor antagonist.
 15. A method according to claim 13, wherein the alpha-2C adrenergic receptor agonist is an agonist selected from the group consisting of epinephrine, norepinephrine, clonidine, oxymetazoline, guanabenz, UTK14304, BHTY933 and combinations thereof.
 16. A method according to claim 14, wherein the alpha-2C adrenergic receptor antagonist is an antagonist selected from the group consisting of yohimbine, prazosin, ARC 239, rauwolscine, idazoxan, tolazoline, phentolamine and combinations thereof.
 17. A method according to claim 12, wherein the predetermined response to the agonist or antagonist is correlated to adenyly cyclase, or MAP kinase activity or inositol phosphate levels. 